Degradation of bisphenol A (BPA, 0.5 L, 30 mg L−1) was studied by photo-Fenton treatment, while Fenton reagents were variables. The efficiency of the degradation process was evaluated by the reduction of total organic carbon (TOC), the biochemical oxygen demand (BOD), and toxicity. For toxicity analysis, bacterial methods were found infeasible, but the in vitro assay of VERO cells culture was successfully applied. Experiments according to a 22 design of experiments (DOE) with star points and three center points for statistical validity allowed selecting those process conditions (Fe(II) and H2O2 load) that maximized the process performance. Photo-Fenton process effectively eliminated BPA and partly degraded its by-products (residual TOC <15 %) under substoichiometric H2O2 dose (100.62 mg L−1) and at least 4 mg L−1 Fe(II), after a 90-min treatment. All treated samples were at least partially biodegradable. The cytotoxic concentration (LD50) of BPA for VERO cells was 7 mg L−1. With small H2O2 amount (15.24 mg L−1), only low BPA mineralization (TOC = 92 %) was attained. Toxicity was also detected to 50 % of cellular mortality even at long reaction times. However, 40.25 mg L−1 of H2O2 decreased residual TOC to 70 % while cell mortality decreased down to 25 %. With more H2O2, the residual TOC decreased down to 15 % but cell mortality remained within the 20–25 % level. Photo-Fenton increased the biodegradability and reduced the toxicity of the studied sample.