The microtiter dish biofilm formation assay is a valuable tool for studying the early stages of biofilm formation, particularly in bacterial systems. Biofilms are communities of microbes attached to surfaces, characterized by an extracellular matrix that provides structure and protection. These biofilms exhibit resistance to antimicrobial agents and are prevalent in various environments. The microtiter dish assay, while not suitable for mature biofilm formation, effectively identifies factors involved in biofilm initiation, such as flagella, pili, adhesins, and enzymes. It has been used to study a wide range of microbes, including *Pseudomonas aeruginosa*, *Vibrio cholerae*, *Escherichia coli*, *Staphylococcus*, *Enterococcus*, *Mycobacteria*, and fungi. The assay measures biofilm formation using crystal violet staining and is flexible, low-cost, and high-throughput, making it ideal for genetic screens and testing multiple strains under different conditions. The method can be adapted for various microbial species, with optimal conditions varying based on the microbe. The protocol includes recommendations for multiple replicates and controls to ensure reliable results.The microtiter dish biofilm formation assay is a valuable tool for studying the early stages of biofilm formation, particularly in bacterial systems. Biofilms are communities of microbes attached to surfaces, characterized by an extracellular matrix that provides structure and protection. These biofilms exhibit resistance to antimicrobial agents and are prevalent in various environments. The microtiter dish assay, while not suitable for mature biofilm formation, effectively identifies factors involved in biofilm initiation, such as flagella, pili, adhesins, and enzymes. It has been used to study a wide range of microbes, including *Pseudomonas aeruginosa*, *Vibrio cholerae*, *Escherichia coli*, *Staphylococcus*, *Enterococcus*, *Mycobacteria*, and fungi. The assay measures biofilm formation using crystal violet staining and is flexible, low-cost, and high-throughput, making it ideal for genetic screens and testing multiple strains under different conditions. The method can be adapted for various microbial species, with optimal conditions varying based on the microbe. The protocol includes recommendations for multiple replicates and controls to ensure reliable results.