Freshwater ecosystems are among the most vulnerable to anthropogenic stressors, with increasing concern over emerging contaminants that may affect aquatic organisms and compromise overall ecosystem health. Among these, substances with teratogenic potential represent an ecotoxicological concern that remains largely unregulated under the current Water Framework Directive (2000/60/EC), despite growing evidence of their biological impact. This study explores the use of biological tools to assess both the ecological status and teratogenic risk in eight riverine systems of Central Italy. Two complementary indicators were applied: the Intercalibration Common Metric Index (ICMi), derived from benthic diatom assemblages as a proxy for ecological quality, and the Teratogenic Risk Index (TRI), based on morpho-functional responses in Hydra vulgaris. Results revealed a wide spatial variability in both ecological status (ICMi ranging from poor to high) and TRI values. Several sites showed elevated teratogenic risk even in the absence of ecological degradation, with some classified as ecologically “good” presenting marked teratogenic effects and thus suggesting the presence of bioactive contaminants undetected by conventional monitoring. Diatom assemblages reflected natural and anthropogenic gradients, with motile and tolerant taxa dominating impacted sites, while low-profile species were more abundant under better environmental conditions. The lack of correlation between ecological quality and teratogenic risk highlights a critical gap in current assessment frameworks. These findings support the need to broaden environmental monitoring to include early warning bioindicators for pollutants currently outside regulatory control. Such an integrated approach can enhance the ability to detect significant ecotoxicological pressures and contribute to more effective strategies for ecosystem protection and regulatory innovation, within a One Health approach.