The objective of this study was to assess the potential role of aquatic biofilms as natural reservoirs for Blastocystis. For this purpose, surface water (n = 4) and biofilm samples (n = 8) were collected from a stream nearby an urban area characterized by limited sanitation infrastructure and a high prevalence of Blastocystis in humans. Blastocystis cysts were detected in three of the four water samples and seven of the eight biofilm samples using fluorescence microscopy. Furthermore, viable cysts were identified exclusively in biofilm samples (five of the eight), while no live cysts were detected in water samples. These findings indicate that aquatic biofilms provide a habitat where Blastocystis cysts can adhere and remain viable, potentially contributing to their environmental accumulation. In addition, molecular characterization of the five isolates identified subtypes ST8 (allele 21) and ST3 (allele 36). This study is the first to report the detection and identification of viable Blastocystis subtypes in aquatic biofilms. The analysis of biofilms by fluorescence microscopy, as demonstrated here, offers a promising approach for monitoring Blastocystis and could serve as an alternative to traditional water sampling methods.