The development of suitable biomarker-based microbioassays with model species with ecological relevance would help increase the cost-efficiency of routine environmental monitoring and chemical toxicity testing. The anti-inflammatory drug diclofenac has been widely reported in the environment but ecotoxicological data are scarce. The aim of this work is to assess the acute and chronic sublethal toxicity of diclofenac in relevant taxa of aquatic and riparian ecosystems (the fish Danio rerio and the fern Polystichum setiferum). Reliable biomarkers of cell viability (mitochondrial activity), plant physiology (chlorophyll), growth (DNA content) or oxidative damage (lipid peroxidation) were assessed as sensitive endpoints of toxicity. DNA quantification shows that diclofenac induces acute lethal phytotoxicity at 24 and 48 h (LOECs 30 and 0.3 μg l−1, respectively). Hormetic effects in mitochondrial activity in spores of Polystichum setiferum mask lethality, and adverse effects are only observed at 48 h (LOEC 0.3 μg l−1). In chronic exposure (1 week) LOEC for DNA is 0.03 μg l−1. Mitochondrial activity shows a strong hormetic stimulation of the surviving spore population (LOEC 0.3 μg l−1). Little changes are observed in chlorophyll autofluorescence (LOEC 0.3 μg l−1). A very short exposure (90 min) of zebrafish embryos induces a reduction of lipid peroxidation at 0.03 μg l−1. Environmental concentrations of diclofenac can be deleterious for the development of significant populations of sensitive individuals in aquatic and riparian ecosystems.