Purpose: Insufficient teaching of clinical microbiology, often caused by limited resources in medical schools, might be a reason for inaccurate diagnosis and treatment of infectious diseases by doctors. The purpose of this study is to develop and assess a multimedia self‐learning tool (MSLT) for clinical microbiology course. Methods: We developed the MSLT based on existing self-directed learning tools. This tool was used by second-and third-year medical students. We randomly assigned 67 participating students to two groups: one (29) with lectures only and the other (38) with the MSLT only. We conducted pre- and post-tests. Results: There are no differences in the pre- and post-test scores between the lecture group and the MSLT group in knowledge of bacterial classification, understanding of infectious diseases, proper use of laboratory tests, and proper selection of antimicrobials. However, post-test scores were significantly higher in both groups. Conclusion: The MSLT was found to be as equally effective as lectures, at least, test scorewise. Teachers could use either this tool alone or combined with conventional lectures to improve and enhance teaching in clinical microbiology. The results shed new insights into the possibility of introducing new teaching methods in clinical microbiology for future medical education.