Metals and metalloids are persistent environmental pollutants with the potential for bioaccumulation, posing significant health risks, including genotoxicity. These contaminants are prevalent in industrial and agricultural runoff. This study utilizes Trachemys dorbigni, an aquatic reptile, as a bioindicator to assess environmental contamination by metals and metalloids in both rural and urban settings in Pelotas, Rio Grande do Sul, Brazil. We captured specimens using pit-type traps with barriers (fyke nets), dividing them into two groups: 15 from a rural area and 15 from an urban area. Each animal underwent physical evaluations, and biometric data (weight, total carapace length and width) were recorded to calculate body condition indices. Biological samples were collected via manual restraint, with blood samples drawn from the supraoccipital venous sinus and linear carapace fragments obtained through manual scraping. Water samples from each location were also analyzed. Using atomic absorption spectrophotometry, concentrations of Al, As, Cd, Cr, Cu, Fe, Hg, Mn, Ni, Pb and Zn were measured in the water, carapace fragments, and blood samples. Cellular damage was assessed through flow cytometry and microscopy, examining erythrocyte disruption, reactive oxygen species, membrane fluidity, DNA fragmentation and micronucleus formation. Urban area samples showed concentrations of As, Cd, Cu, Fe, Hg and Ni exceeding national standards set by the Conselho Nacional do Meio Ambiente (CONAMA), with rural areas also showing elevated levels of As, Cd, Hg and Ni. Biometric analysis revealed that rural reptiles had significantly higher weight and carapace dimensions, whereas urban tortoises displayed a higher body condition index and significantly elevated blood levels of Al, Cr, Ni, Pb and Zn. The urban tortoises also exhibited higher concentrations of all tested metal(loid) in carapace samples (p < 0.05) and more pronounced cellular damage (p < 0.05), highlighting severe bioaccumulation and associated deleterious effects. Elevated reactive oxygen species levels were noted in rural specimens. This study underscores the impact of water degradation and metal(loid) pollution in urban environments on T. dorbigni, suggesting that carapace tissue analysis can serve as a chronic exposure indicator to these harmful contaminants.