This study aimed to detect and identify emerging pathogenic yeasts in surface waters from two Lagoon Systems in Rio de Janeiro, Brazil, by polyphasic taxonomy. The monitoring of potentially pathogenic fungi in aquatic environments, especially in regions impacted by human actions, is highlighted in this study as one of the axes of the One Health approach. Water samples were collected, and after isolation, fungi were identified by polyphasic taxonomy, including MALDI-TOF–MS, which is also used for bacteria identification. Our results describe the physicochemical parameters of the surface water of these lagoons and reveal a significant diversity of yeast species, some of which are known to exhibit pathogenic potential and resistance to common antifungal treatments, as well as bacteria with resistance profiles to several currently used antibiotics. We conclude that Jacarepaguá Lagoon is the most impacted compared to Piratininga Lagoon; however, we highlight the urgent need for targeted interventions to mitigate pollution in both Lagoons, reinforcing the value of long-term surveillance in both ecosystems. We believe that monitoring emerging species is as critical as analyzing traditional water quality parameters. Early detection of resistant or invasive pathogens in aquatic ecosystems can prevent disease outbreaks, protect biodiversity, and mitigate public health risks, preventing loss of life, and optimizing public healthcare system expenditures.