A new microbiological method was developed for analysis of luliconazole cream using Candida albicans as test organism. The diffusion agar method was optimized by using different media organisms and conditions. The purpose of this research is to establish a microbiological analytical method for the estimation of the pure and pharmaceutical dosage type (cream) of luliconazole and to validate the method as per the ICH guidelines. As a comparative tool for luliconazole estimation, high performance liquid chromatography (HPLC) was selected. Acceptable linearity (0.9965), accuracy (percent Relative Standard Deviaton ˂2) and accuracy (mean percent recovery = 100.12) were seen by a prospective validation of the process. The statistics of the microbiological and HPLC methods were correlated with the student t-test, and there was a considerable link among both the luliconazole content determined by both methods. The developed microbiological analytical method provides a true indication of biological activity and can be used for routine analysis of the quality control of luliconazole in drug formulations. The microbiological approach is a procedure which does not make use of organic solvents and advanced equipment for its study poses little worry for chemical waste and is cost-effective in contrast with instrumental methods such as HPLC and UV-spectrophotometry. According to the literature, when measured using a physicochemical procedure, measuring the content of an antimicrobial agent by its potency will produce altered result.