<p>Urban rivers worldwide are increasingly exposed to multiple stressors from industrial, agricultural, and domestic activities; however, sensitive indicators to assess their ecological status remain limited. This study evaluated the potential of natural biofilms as bioindicators of environmental quality in two impacted river basins in Argentina: the Luján and Reconquista rivers. Biofilm and water samples were collected from upstream, urban, and industrial sites, and from controlled outdoor ponds used as a control. We analysed water physicochemical parameters, biofilm community composition, oxidative stress biomarkers (reduced glutathione, GSH; catalase, CAT; glutathione S-transferase, GST; and thiobarbituric acid reactive substances, TBARS), and extracellular enzyme activities (alkaline phosphatase, β-glucosidase). Results revealed clear water quality gradients, with downstream Luján sites and the mid-Reconquista showing severe deterioration, nutrient enrichment, hypoxia, and heavy metal exceedances. Biofilm communities mirrored these conditions: diverse and balanced assemblages dominated reference sites, while degraded ones showed reduced richness and tolerant taxa dominance. Biomarker responses also displayed consistent alterations, with high CAT activity (11,87 mmoles H₂O₂ cons x min⁻¹ × mg prot⁻¹ in L3 and 16,04 in R2), reduced GSH (0,007 mmoles GSH x g ww⁻¹ in R2), and imbalances in GST and TBARS, indicating enhanced oxidative stress. Multivariate analyses integrated these datasets, consistently separating less-impacted from highly degraded sites. Overall, biofilms proved to be sensitive and cost-effective indicators, integrating structural and functional responses to contamination gradients, and offering a robust tool for monitoring urban rivers.</p>