Diclofenac, a nonsteroidal anti-inflammatory drug widely used worldwide, has been detected in waterbodies at concentrations ranging from ng L⁻¹ to µg L⁻¹. Although diclofenac is not a persistent compound, aquatic organisms may be exposed to this drug for extended periods due to its incorporation into the environment by continuous release from hospitals and municipal discharges. This study aimed to evaluate the toxic effects of diclofenac on the microalga Pseudokirchneriella subcapitata, the cladoceran Daphnia curvirostris, and zebrafish embryos (Danio rerio). Toxicity bioassays for the microalga were performed according to the OECD 201 protocol with diclofenac concentrations of 0, 6.25, 12.5, 25, 50, 75, and 100 mg L⁻¹. For the determination of acute toxicity in the cladoceran (48 h), concentrations of 0, 10, 20, 30, 40, 50, and 60 mg L⁻¹ were tested; in subchronic bioassays, the effect of the drug on the reproductive parameters of D. curvirostris was determined for 21 days with sublethal concentrations of 10.3, 14.4, 17.2, and 21.3 mg L⁻¹. Toxicity bioassays on zebrafish embryos were performed according to the OECD 236 protocol, using concentrations of 0, 1, 2, 4, 6, 8, and 10 mg L⁻¹ of diclofenac. The results confirmed the toxic effects of the drug. The IC₅₀ for the microalga was 16.57 mg L⁻¹, while the LC₅₀ for D. curvirostris and D. rerio was 32.29 and 6.27 mg L⁻¹, respectively. In the microalga, chlorophyll-a and carotenoids increased at a concentration of 3.62 mg L⁻¹ of diclofenac; however, chlorophyll-b decreased at the highest drug concentration (13.51 mg L⁻¹). Protein and lipid concentrations in P. subcapitata exposed to all concentrations were higher than in the control. Chronic diclofenac exposure did not affect the survival of D. curvirostris; however, the cumulative progeny and number of clutches significantly decreased, and the age of first reproduction was delayed at all drug concentrations. Protein concentration in D. curvirostris hatchlings was higher at all diclofenac concentrations; in contrast, the amount of lipids and carbohydrates decreased significantly. In D. rerio, the hatching rate decreased by 40, 51.6, and 80% at concentrations of 6, 8, and 10 mg L⁻¹ diclofenac, respectively, and exposure to the drug caused lethal effects such as coagulation at 24 and 48 hpf; sublethal effects such as edema and curved tail were also observed at concentrations of 2 to 10 mg L⁻¹, and the effects increased with increasing concentration up to 144 hpf. The results demonstrate the vulnerability of aquatic organisms to the toxic effects of diclofenac, suggesting that discharging it into water bodies should be regulated to prevent potential ecological impacts on the various trophic levels of freshwater biota.