Yeasts are unicellular fungi widespread in the environment, and studies suggest that there is a positive correlation between yeast and polluted aquatic environments. The aim of this study was to analyze the diversity and resistance of yeasts isolated from water samples collected along the Arroio Dilúvio in Porto Alegre to antifungals. Yeast isolates from the Arroio Dilúvio were subjected to susceptibility assays against antifungals using the minimum inhibitory concentration (MIC) test, and amplification of the ITS1-5.8S-IT2 region; in addition, the ITS-5.8S region was sequenced for some of the isolates. The amplification product was subjected to PCR-RFLP, and the restriction profile allowed the construction of a dendrogram. Susceptibility tests showed a high prevalence of resistance to azole antifungals, where 16.8% of the isolates had a resistance profile to amphotericin B. The sequence analysis allowed the identification of Candida species, including potentially pathogenic species, and species of the Debaryomyces spp. The resistance to antifungals in yeasts isolated from Arroio Dilúvio reinforces the importance of studies of environmental microbiota, and indicates that environmental degradation influences the phenotype displayed.