Purpose: The current study investigated how clinical microbiology courses are conducted at the undergraduate nursing level in South Korea to identify the perceptions of clinical nurses regarding their knowledge and the clinical importance of microbiology and further assess their educational needs. Methods: Curricula presented on the websites of 202 undergraduate nursing institutions were reviewed. In addition, a survey assessing the knowledge and importance of clinical microbiology perceived by nurses was conducted online. A total of 150 nurses participated in the survey. Data were analyzed using descriptive statistics, correlation coefficients, t-tests, analysis of variance, Borich Needs Assessment Model, and Locus for Focus Model. Results: The results indicated that 90% of undergraduate nursing institutions offer clinical microbiology courses under various subject names. The perceived knowledge and importance of clinical microbiology were correlated; however, knowledge was consistently lower than the perceived importance of clinical biology among nurses. The structure and proliferation of novel viruses ranked highest in the educational needs of nurses based on both the Borich Needs Assessment Model and Locus for Focus Model. Conclusion: Based on the findings, this study suggests educational opportunities for clinical microbiology in hospital settings to reduce discrepancies between knowledge and the importance of clinical microbiology faced by nurses.