<p>The widespread occurrence of the fluoroquinolone antibiotic ofloxacin (OFL) in freshwater ecosystems poses an emerging threat to non-target aquatic macrophytes. This study investigated the concentration-dependent phytotoxic effects of OFL (0.25–10.0&#xa0;µg L⁻¹) on <i>Azolla pinnata</i> by integrating biochemical biomarkers with chlorophyll <i>a</i> fluorescence (OJIP) analysis. OFL exposure caused progressive morphological deterioration, reduced relative growth rate, and significant chlorophyll (Chl) loss, accompanied by elevated H₂O₂ and malondialdehyde accumulation, indicating enhanced oxidative stress. Superoxide dismutase and catalase activities showed an initial stimulation at lower concentrations followed by suppression at higher doses, reflecting antioxidant system failure under severe stress. ChlF and JIP-test analyses revealed impaired PSII photochemistry, reduced electron transport, reaction centre inactivation, and increased energy dissipation. Multivariate analyses confirmed strong negative associations between oxidative stress and photosynthetic performance. Overall, OFL disrupted redox homeostasis and photosynthetic efficiency, demonstrating that <i>A. pinnata</i> is a sensitive bioindicator for assessing antibiotic-induced ecological risks in freshwater ecosystems.</p> Graphical abstract <p></p>