This study assessed the impact of untreated wastewater discharge in the Danube River on four native fish species: barbel (Barbus barbus), vimba bream (Vimba vimba), perch (Perca fluviatilis), and white bream (Blicca bjoerkna). Biomarkers of exposure and effect were evaluated, including metal and metalloid bioaccumulation in gills, liver, and gonads, DNA damage (comet assay), chromosomal abnormalities (micronucleus assay), liver enzyme activities (ALT, AST), and erythrocyte maturation. White bream showed the highest genotoxic damage (TI% = 22.57), particularly in liver tissue, indicating high sensitivity to pollution. Perch had elevated DNA damage in blood (TI% = 22.69) and strong biomarker responses, likely due to its predatory behavior. Barbel displayed notable DNA damage in gills (TI% = 30.67) and liver (TI% = 20.35), aligning with sediment exposure due to its benthic habits. Vimba bream had the lowest responses, possibly reflecting reduced exposure or resilience. Element accumulation varied across tissues and species, with perch showing the highest overall levels. Hepatic enzyme activities (highest values: ALT = 105.69 in barbel; AST = 91.25 in white bream) and changes in erythrocyte profiles supported evidence of physiological stress. Integrated Biomarker Response (IBR) analysis identified white bream as the most sensitive species, followed by perch and barbel. These results emphasize the value of multi-species biomonitoring and the importance of species-specific traits in freshwater ecotoxicology.