The microtiter dish biofilm formation assay is a widely used method for studying biofilm formation in bacteria and fungi. Biofilms are communities of microbes attached to surfaces, which are common in medical, industrial, and natural environments. Biofilm-forming microbes are surrounded by an extracellular matrix and have a characteristic architecture. They are also resistant to antimicrobial agents and immune system effectors. The microtiter dish assay is useful for studying the early stages of biofilm formation and has been used for both bacterial and fungal biofilms. It uses static, batch-growth conditions and does not allow for the formation of mature biofilms. However, it is effective at identifying factors required for biofilm initiation and genes involved in extracellular polysaccharide production. Biofilms grown in microtiter dishes develop some properties of mature biofilms, such as antibiotic tolerance. The assay involves growing a microbial culture, diluting it, and adding it to a microtiter dish. The dish is incubated, and the biofilm is stained with crystal violet. The biofilm is then rinsed, dried, and quantified using a plate reader. The extent of biofilm formation is measured by the amount of crystal violet retained. This assay is simple, cost-effective, and high-throughput, making it suitable for genetic screens and testing biofilm formation under various conditions. It has been used for a wide range of microbes, including pseudomonads, Vibrio cholerae, Escherichia coli, staphylococci, enterococci, mycobacteria, and fungi. The protocol described here focuses on Pseudomonas aeruginosa, a model organism for studying biofilm formation. The assay has been modified for use with various microbial species, with optimal conditions determined empirically for each microbe. Multiple replicates and controls are recommended for accurate results.The microtiter dish biofilm formation assay is a widely used method for studying biofilm formation in bacteria and fungi. Biofilms are communities of microbes attached to surfaces, which are common in medical, industrial, and natural environments. Biofilm-forming microbes are surrounded by an extracellular matrix and have a characteristic architecture. They are also resistant to antimicrobial agents and immune system effectors. The microtiter dish assay is useful for studying the early stages of biofilm formation and has been used for both bacterial and fungal biofilms. It uses static, batch-growth conditions and does not allow for the formation of mature biofilms. However, it is effective at identifying factors required for biofilm initiation and genes involved in extracellular polysaccharide production. Biofilms grown in microtiter dishes develop some properties of mature biofilms, such as antibiotic tolerance. The assay involves growing a microbial culture, diluting it, and adding it to a microtiter dish. The dish is incubated, and the biofilm is stained with crystal violet. The biofilm is then rinsed, dried, and quantified using a plate reader. The extent of biofilm formation is measured by the amount of crystal violet retained. This assay is simple, cost-effective, and high-throughput, making it suitable for genetic screens and testing biofilm formation under various conditions. It has been used for a wide range of microbes, including pseudomonads, Vibrio cholerae, Escherichia coli, staphylococci, enterococci, mycobacteria, and fungi. The protocol described here focuses on Pseudomonas aeruginosa, a model organism for studying biofilm formation. The assay has been modified for use with various microbial species, with optimal conditions determined empirically for each microbe. Multiple replicates and controls are recommended for accurate results.