Although antibiotics have been increasingly used and detected in natural samples, their ecotoxicological effects on aquatic wildlife are not yet extensively studied. Considering the environmental threat posed by the biological activity of antibiotics it is quite relevant to assess the resulting impact, especially on sub-lethal endpoints. As such, this study evaluated the effects of ciprofloxacin on Pseudokirchneriella subcapitata and Lemna minor growth, on the survival and reproduction of Daphnia magna and on Gambusia holbrooki survival. The risks associated with ciprofloxacin effects on non-target organisms were quantified through the calculation of the PEC/PNEC ratio. Overall, the toxicity values obtained (at the mg L−1 level) were higher than the environmental concentrations. P. subcapitata and L. minor were more sensitive under short-term exposures than D. magna and G. holbrooki. No acute toxicity was observed for fish. The chronic assay with D. magna evidenced that long term exposures to lower concentrations of this antibiotic induced impairments on its life-history parameters. Such outcome may pre-empt potential damages on the long-term maintenance of natural populations continuously exposed to the input of antibiotics. Indeed, the PEC/PNEC ratios showed that ciprofloxacin represents a risk for the most sensitive aquatic organisms, since the defined threshold of an acceptable risk was considerably surpassed.