In this study we investigated the influence of preparation of the bacterial inoculum for a microdilution susceptibility test, e.g., the effect of its optical density, on assessment of the minimum inhibitory concentrations (MIC). The approach employed in the majority of microdilution susceptibility studies is use of the same optical density for preparation of inoculums for different bacterial strains. In the present work, this approach was questioned by determining the ratio between the optical density and the number of bacteria in cultures. We also investigated whether the number of bacteria in inoculums can affect assessment of the MIC value for two antibiotics of broad spectra, rifampicin and streptomycin. The study was performed on four Gram-positive and four Gram-negative bacteria (ATCC collection) commonly used to investigate antimicrobial potential. The ratio between the optical density and number of bacteria in cultures was determined for each strain, and a strong linear correlation was detected. However, it was evident that different bacteria have different cell numbers at the same OD600 value. Based on the obtained results, inoculums for selected strains were prepared to obtain final cell numbers of 103, 104, 105 and 106 /well in the microdilution assay. Two different approaches were used in determining the MIC for rifampicin and streptomycin: approximation of MIC with IC90 and the resazurin reduction assay. Our results indicated that the ratio between optical density and cell numbers is not constant and use of the same OD for inoculums for all strains can therefore lead to misinterpretation of the MIC values. We also observed influence of cell numbers in inoculums in determination of MIC values. For both approaches used (approximation of MIC with IC90 and the resazurin reduction assay), the same trend was detected: antibiotics had the highest potency in experiments with the lowest bacteria cell number (103/well). The lowest cell number (103/well) is not recommended, as it can lead to false susceptibility results and to partial reduction of resazurin, which further complicates MIC determination. A final cell number of 104/well can therefore be recommended as optimal.