Natural deep eutectic solvents (NADESs) are promising alternatives to antibiotics due to their antimicrobial properties. These solvents are derived from natural sources and offer advantages such as low volatility, low flammability, ease of preparation, and low cost. They are used in various applications, including the extraction of biomolecules and the preparation of cosmetics. NADESs have shown significant antibacterial and antifungal activities against a range of microorganisms, making them a potential solution to the growing problem of antibiotic resistance. NADESs are prepared using various methods, including heating and stirring, freeze-drying, evaporation, grinding, and ultrasound-assisted or microwave-assisted synthesis. Different techniques are used to evaluate their antimicrobial activity, such as the disc diffusion method, agar and broth dilution techniques, Microtox assay, drop plate method, and FTIR-based biological assays. These methods help determine the minimum inhibitory concentration (MIC) and assess the toxicity of NADESs. The disc diffusion method is commonly used to evaluate antimicrobial activity, where a filter paper disc impregnated with the testing substance is placed on an agar plate. The inhibition zone around the disc indicates the effectiveness of the substance. The agar dilution method involves applying a predetermined inoculum of microorganisms to nutrient agar plates with varying concentrations of the tested antimicrobial agent. The broth dilution method involves growing bacteria in liquid nutrient medium with increasing concentrations of the antimicrobial agent. The Microtox assay uses bioluminescent bacteria to determine the toxicity of substances. The drop plate method involves serial dilutions of the tested substance and drop plating on agar. FTIR-based biological assays use infrared spectroscopy to analyze changes in the molecular structure of microorganisms after exposure to NADESs. Electron microscopy techniques help visualize the effects of NADESs on microbial cells, providing insights into their mechanism of action. NADESs have shown strong antimicrobial activity against both gram-positive and gram-negative bacteria, as well as fungi. The effectiveness of NADESs is influenced by factors such as the type of hydrogen bond donor and acceptor, the chemical composition of the starting materials, and the method of preparation. Organic acid-based NADESs have demonstrated the strongest antimicrobial activity due to their ability to lower pH and disrupt microbial membranes. NADESs have potential applications beyond antimicrobial activity, including the extraction and purification of virus-like particles, the treatment of biofilms, and the cytotoxicity of tumor cells. Their biodegradability and low toxicity make them promising alternatives to traditional antibiotics and other chemical solvents. However, further research is needed to fully understand their biological activities and to identify the most effective NADESs for large-scale industrial use.Natural deep eutectic solvents (NADESs) are promising alternatives to antibiotics due to their antimicrobial properties. These solvents are derived from natural sources and offer advantages such as low volatility, low flammability, ease of preparation, and low cost. They are used in various applications, including the extraction of biomolecules and the preparation of cosmetics. NADESs have shown significant antibacterial and antifungal activities against a range of microorganisms, making them a potential solution to the growing problem of antibiotic resistance. NADESs are prepared using various methods, including heating and stirring, freeze-drying, evaporation, grinding, and ultrasound-assisted or microwave-assisted synthesis. Different techniques are used to evaluate their antimicrobial activity, such as the disc diffusion method, agar and broth dilution techniques, Microtox assay, drop plate method, and FTIR-based biological assays. These methods help determine the minimum inhibitory concentration (MIC) and assess the toxicity of NADESs. The disc diffusion method is commonly used to evaluate antimicrobial activity, where a filter paper disc impregnated with the testing substance is placed on an agar plate. The inhibition zone around the disc indicates the effectiveness of the substance. The agar dilution method involves applying a predetermined inoculum of microorganisms to nutrient agar plates with varying concentrations of the tested antimicrobial agent. The broth dilution method involves growing bacteria in liquid nutrient medium with increasing concentrations of the antimicrobial agent. The Microtox assay uses bioluminescent bacteria to determine the toxicity of substances. The drop plate method involves serial dilutions of the tested substance and drop plating on agar. FTIR-based biological assays use infrared spectroscopy to analyze changes in the molecular structure of microorganisms after exposure to NADESs. Electron microscopy techniques help visualize the effects of NADESs on microbial cells, providing insights into their mechanism of action. NADESs have shown strong antimicrobial activity against both gram-positive and gram-negative bacteria, as well as fungi. The effectiveness of NADESs is influenced by factors such as the type of hydrogen bond donor and acceptor, the chemical composition of the starting materials, and the method of preparation. Organic acid-based NADESs have demonstrated the strongest antimicrobial activity due to their ability to lower pH and disrupt microbial membranes. NADESs have potential applications beyond antimicrobial activity, including the extraction and purification of virus-like particles, the treatment of biofilms, and the cytotoxicity of tumor cells. Their biodegradability and low toxicity make them promising alternatives to traditional antibiotics and other chemical solvents. However, further research is needed to fully understand their biological activities and to identify the most effective NADESs for large-scale industrial use.