Understanding the behavioral pathways of xenobiotics in the environment allows for the assessment of the risk to humans resulting from their presence. The aim of the study was to assess whether photo-induced degradation of cefazolin is linked to phototoxic effects on living cells. Cefazolin solutions were exposed to UV-Vis radiation, and then quantitative analysis was performed using the validated UPLC-MS/MS method. The obtained data indicate that the photodegradation of cefazolin proceeds according to first-order kinetics and results in the formation of four main products. To understand the environmental modification on the toxicity of cefazolin and its photodegradation products to human health, we used a human skin culture and animal models. Both in vitro and in vivo studies help assess not only the toxicity of the original drug but also the potential ecological risks posed by its degradants. Considering the potential species-specific ecotoxicity of products for biota, it was necessary to use a set of organisms representing different trophic levels. The bioindication tests using Thamnocephalus platyurus and Daphnia magna as test organisms have shown similar EC50 values after 48 h of incubation (≈40 µg/mL). The luminescence inhibition test (LumiMara), based on the measurement of luminescence quenching of saltwater and freshwater bacterial strains, showed increased toxicity of the tested mixtures obtained during irradiation at different time. The highest toxicity was observed for saltwater strains #2 Photobacterium phosphoreum (NCIMB 30267) for all irradiated solutions (MTC=0.030 µg/mL, after 8 h exposure), whereas in the case of non-irradiated solutions it was freshwater strain #11 Photorhabdus asymbiotica (NCIMB 30276), showing the lowest MTC values (12.913 µg/mL). In vivo studies have shown that cefazolin and its photodegradation products have a pronounced effect on the development, motility and cardiac function of Zebrafish larvae, which is manifested by increased mortality (by approx. 10 % for a concentration of 1 µg/mL; 2 h), body deformations (by approx. 0–10 % for a concentration 10 µg/mL and by 0 ̶ 20 % for 50 µg/mL compared to the control), and changes in heart rate (slowing of the heart rate by approx. 5–15 bpm noticeable with longer irradiation times, i.e. 6 and 8 h). Similarly, in vitro cytotoxicity studies using a cell proliferation assay showed increased cytotoxicity of the mixtures obtained after the longest exposure period compared to the non-irradiated antibiotic. The experimental results are consistent with the in silico data, which indicate increased toxicity of photodegradants, especially their cardiotoxic and cytotoxic potential. The performed analyses clearly indicate a higher toxicity of the tested mixtures compared to pure cefazolin in relation to living organisms.