Introduction In this master's thesis, the need for a more profound understanding of nursing students' attitudes toward microbiology and its role in their daily nursing practice is emphasized. Understanding microbiology becomes essential for nurses in making crucial decisions regarding infection prevention, antibiotic management, safe patient and equipment handling, and the proper application of aseptic techniques. Research Objective The objective of this study was to assess the attitudes of undergraduate and graduate nursing students at the Croatian Catholic University regarding the significance of microbiology in the daily work of nurses and technicians. Additionally, it aimed to evaluate their knowledge of microbiology, its application in nursing practice, and their motivation for further development of their microbiological diagnostic skills. Methods Participants completed an online survey that included questions about their educational background, work experience, microbiology education, types of microbiological samples encountered in their work, their responsibilities in sample preparation, collection, and submission, and their interest in further education in microbiology. Results The study included a total of 71 participants. The majority of participants (49.3%) completed their undergraduate studies, while 21.1% completed graduate studies. A smaller number of participants, 1.4%, had a postgraduate degree. Additionally, 28.2% of participants had a secondary vocational education. Regarding years of work experience, the majority of respondents (38.0%) had less than one year of experience, while 30.9% of participants had 1 to 5 years of experience. The group with 6 to 10 years of experience comprised 14.0% of respondents. A smaller number of participants had work experience ranging from 11 to 15 years (4.0%), while 2.8% of participants fell into the categories of experience duration of 16 to 20 years, 26 to 30 years, 31 to 40 years, and over 40 years. Microbiology education was primarily based on lectures and seminars. Most participants did not have access to a microbiology laboratory during their education. In their work, participants actively deal with microbiological samples during patient care. Participants expressed an interest in further continuing education in microbiology. Conclusion This research contributes to a better understanding of the role of microbiology in nursing education. Emphasis was placed on the importance of nurses' knowledge in identifying types of microbiological samples and obtaining targeted samples for microbiological analysis, as well as in preparing patients for sample collection. The study provides guidelines for improving the educational process by incorporating practical work in microbiological laboratories to acquire essential skills in this field.