A number of drugs are constantly released into the environment, making them potentially hazardous pollutants. The presence of antibiotics can cause adverse effects, and unfortunately still little is known about their fate in the environment. We conducted comprehensive ecotoxicity studies, paying attention to the assessment of exposure of living organisms to cephalosporin and mixture of the substance and its photoproducts. The kinetics of photodegradation of cefuroxime axetil were determined, and molecular structure of photoproducts was proposed. The in silico method was used to determine the potential ecotoxicity of products in relation to substrate. Environmental impact was investigated using ecotoxicological tests (Thamnotoxkit F, Daphtoxkit F, LumiMARA) and in vivo studies (MTT, Danio rerio). In silico data suggested that cefuroxime and its degradants had rather similar toxicity, while ecotoxicological analyzes and in vitro cytotoxicity studies indicated greater toxicity of the irradiated mixture. The LumiMARA results showed growth inhibition of all tested freshwater bacteria. Studies on zebrafish have shown that mixtures are significantly more toxic compared to the substrate. The obtained results indicate the presence of new compounds formed under the influence of light, which show significant toxicity in the aquatic environment.