Background: The increasing occurrence of emerging contaminants, such as pharmaceuticals, in freshwater ecosystems poses a significant environmental concern. Metformin (MET) is a widely prescribed antidiabetic for type-II diabetes mellitus treatment, with growing off-label uses. Its continuous use, combined with limited removal efficiency in conventional wastewater treatment plants, results in its persistent release into surface waters, posing potential risks to aquatic wildlife and ecosystem stability. This study evaluated the short-term ecotoxicological effects of MET on standard freshwater species, integrating physiological responses and biochemical biomarkers. Methods: All bioassays followed OECD guidelines: growth inhibition assays for Raphidocelis subcapitata (3 days; 0 to 1000 mg MET/L) and Lemna minor (7 days; 0 to 200 mg MET/L), acute immobilization assays for Daphnia magna (2 days; 0 to 120 mg MET/L) and fish embryo acute toxicity test for Danio rerio (4 days; 0 to 3000 mg MET/L). In L. minor, biomarkers of oxidative stress, detoxification, lipid peroxidation, and photosynthetic pigments were quantified. For D. magna and D. rerio, endpoints related to oxidative stress, detoxification, lipid peroxidation, and neurotoxicity were assessed. Results: MET exposure significantly inhibited growth in R. subcapitata (EC₅₀ = 222.4 mg/L) and L. minor (EC₅₀ = 50.8 mg/L) in a concentration-dependent pattern. In D. magna, MET induced acute toxicity (LC₅₀(48h) = 70.64 mg/L; EC₅₀(48h) = 53.67 mg/L). For D. rerio, MET did not seem to affect embryo mortality or hatching rates, and developmental abnormalities remained low (<25%). In L. minor, increased antioxidant and detoxification responses seem to preserve photosynthetic function, although lipid peroxidation has been observed. Moreover, MET induced oxidative stress and neurotoxicity in D. magna and significantly affected antioxidant and detoxification enzymes in D. rerio embryos. Conclusion: These findings emphasise metformin's potential to induce adverse effects across multiple trophic levels, with implications for food web dynamics, water quality, and ecosystem integrity.