The presence of fungi in healthcare settings, including hemodialysis units, represents a significant risk for immunocompromised patients. This study aimed to investigate the occurrence of fungi in the air and water of a hemodialysis unit located in a tertiary public hospital in Maceió, Alagoas, Brazil. Over a period of three consecutive months, monthly air samples were collected and analyzed using the spontaneous sedimentation technique on Petri dishes containing Sabouraud Dextrose Agar (SDA). Simultaneously, water samples (100 mL) were collected from four specific water distribution points and subjected plating on SDA. Fungi were phenotypically identified based on their macroscopic and microscopic characteristics. In total, 498 colony-forming units (CFUs) of fungi were isolated, with 86 CFUs originating from the air and 412 CFUs from the water. Regarding the water samples, a higher concentration of fungal CFUs was observed in the potable water from the supply network (229 CFUs). Unexpectedly, 23 CFUs were identified in the reverse osmosis samples and 11 CFUs in the storage tank, which are post-treatment points where the presence of microorganisms is not desired. The fungus Cladosporium spp. was the most prevalent in both air and water samples, followed by Penicillium spp. in the air and Rhodotorula spp. in the water. These findings underscore the need to implement effective control and monitoring measures for fungi in the hemodialysis unit to ensure patient safety.