The article reviews the use of nucleic acid-based methods to investigate microbial-related cheese quality defects. Cheese quality is significantly influenced by the microbial profile, which can lead to various defects such as aroma and taste issues, biogenic amine formation, gas formation, secondary fermentation, mineral deposition, and cheese pinking. These defects can result from seasonal variations, milk composition, processing parameters, and the presence of non-starter microorganisms. Traditional culture-dependent methods are often biased and slow, whereas molecular techniques provide rapid and accurate detection of specific spoilage microbes, aiding in enhancing cheese quality and reducing economic losses. The review covers various molecular methods, including PCR-based techniques (such as qRT-PCR and next-generation sequencing), denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient electrophoresis (TTGE), single-strand conformation polymorphism (SSCP), and terminal restriction fragment length polymorphism (T-RFLP). These methods are effective in identifying and quantifying specific microbial populations and metabolic pathways, enabling early detection and control of cheese defects. The article also discusses the potential use of next-generation sequencing platforms for cheese industry applications.The article reviews the use of nucleic acid-based methods to investigate microbial-related cheese quality defects. Cheese quality is significantly influenced by the microbial profile, which can lead to various defects such as aroma and taste issues, biogenic amine formation, gas formation, secondary fermentation, mineral deposition, and cheese pinking. These defects can result from seasonal variations, milk composition, processing parameters, and the presence of non-starter microorganisms. Traditional culture-dependent methods are often biased and slow, whereas molecular techniques provide rapid and accurate detection of specific spoilage microbes, aiding in enhancing cheese quality and reducing economic losses. The review covers various molecular methods, including PCR-based techniques (such as qRT-PCR and next-generation sequencing), denaturing gradient gel electrophoresis (DGGE), temporal temperature gradient electrophoresis (TTGE), single-strand conformation polymorphism (SSCP), and terminal restriction fragment length polymorphism (T-RFLP). These methods are effective in identifying and quantifying specific microbial populations and metabolic pathways, enabling early detection and control of cheese defects. The article also discusses the potential use of next-generation sequencing platforms for cheese industry applications.