Background: Hospital-acquired fungal infections are becoming an increasingly significant cause of morbidity, mortality, and healthcare costs. Nurses play a pivotal role in preventing and managing these infections, yet their knowledge levels often remain unevaluated. Objectives: This study aimed to assess the knowledge of nursing staff in educational-medical hospitals in Zahedan, Iran, regarding fungal infections in 2023. Methods: This descriptive, cross-sectional study was conducted among nurses in three educational hospitals (Khatam Al Anbia, Ali ebn Abitaleb, and Bu Ali) in Zahedan. A total of 342 nurses with at least one year of work experience were included using convenience sampling. Data were collected using a two-part questionnaire: A demographic information form and a researcher-made questionnaire assessing knowledge about fungal infections across four domains—epidemiology, prevention, diagnosis, and treatment. Data were analyzed using SPSS software, with a P-value of less than 0.05 considered significant. Results: The mean age of participants was 40.70 ± 8.36 years, with an average work experience of 237.85 ± 133.02 months. Of the 342 nurses, 52.92% were female. The mean total awareness score was 12.63 ± 4.53 out of 25. Overall, 19.30% had very low awareness, 52.05% had low awareness, 26.32% had moderate awareness, and only 2.34% had high awareness. In the epidemiology domain, 69.30% had very low or low knowledge. For prevention, 61.11% showed very low or low awareness. In diagnosis, 61.40% had very low or low knowledge, and in treatment, 63.45% demonstrated very low or low awareness. No significant differences were found based on work experience, gender, age, educational level, or hospital affiliation (P > 0.05). Conclusions: The study reveals a critical knowledge deficit among nursing staff in Zahedan regarding fungal infections. Over 70% of nurses demonstrate low or very low overall awareness, a trend consistent across all evaluated domains. This systemic issue underscores the urgent need for comprehensive, targeted educational interventions, ongoing professional development, and institutional policy changes to enhance nurses' competency in managing fungal infections. Such measures are crucial for improving patient safety, reducing infection rates, and ultimately saving lives.