ISO/IEC 17025 : 2017 requires laboratories to verify the performance of methods before introducing them. ISO 16140-3 : 2021 employs LOD₅₀ (the level of detection for which 50% of tests give a positive result) as an index for evaluating the performance of microbial qualitative methods. While fecal coliforms and Staphylococcus aureus qualitative methods are widely implemented in Japan, their LOD₅₀ values have not been established, making performance evaluation of these methods impossible at the time of introduction. As a result, we looked into estimating the LOD₅₀ values of fecal coliforms and S. aureus qualitative methods to evaluate the index of method performance. The fecal coliforms method had an LOD₅₀ value of 19–31 CFU/mL and the S. aureus method had an LOD₅₀ value of 29–49 CFU/mL by laboratory personnels using samples without food matrix. We estimated LOD₅₀ of six food samples using each method in a single laboratory. The LOD₅₀ values of all foods calculated using the fecal coliforms method were in the range of 14–27 CFU/g and those calculated using the S. aureus method were in the range of 25–48 CFU/g, indicating that these methods did not have low levels of sensitivity detection compared with skill variation. LOD₅₀ values for food samples with the highest or lowest LOD₅₀ in each method were compared among five laboratories. Because the estimated LOD₅₀ values of both the methods were within four times of each laboratory result, which is the acceptable limit specified in ISO 16140-3 : 2021, they were assumed to be suitable values. This study is expected to be utilized as a benchmark in the verification of method performance in each laboratory.