The use of poorly treated water in hemodialysis centers may lead to fungal contamination, which poses a serious threat to immunologically debilitated hemodialysis patients. This study aimed to isolate and identify yeast species in the water of a Brazilian hemodialysis center by using classic microbiological techniques and Raman spectroscopy. For 12 months, a total of 288 water samples were collected from different points of the hemodialysis treatment distribution center. One hundred and forty-six yeast species were isolated and identified in the samples that tested positive for the presence of yeasts such as Candida parapsilosis (100 isolates, or 68.50%), C. guilliermondii (17 isolates, or 11.65%), Rhodotorula mucilaginosa (23 isolates, or 15.75%), R. glutinis (three isolates, or 2.05%), and Trichosporon inkin (three isolates, or 2.05%). Yeast susceptibility to the antifungal fluconazole was also assayed. Only two C. guilliermondii isolates were resistant to fluconazole: the minimal inhibitory concentrations were higher than 64 μg/mL. The different yeast species present in the water of a Brazilian hemodialysis center call for more effective water disinfection procedures in this unit. Raman spectroscopy is an excellent tool to identify yeast species and is potentially applicable in routine water monitoring in hemodialysis units.