Urban, hospital and pharmaceutical industry wastewater effluents are among the main sources of antibiotics' and other antimicrobial agents' contamination in soil and water ecosystems, especially in countries where wastewater reuse is applied. Ampicillin (AMP), which is the first semi-synthetic broad spectrum penicillin that was released on the market in 1961 has been detected in treated wastewater effluents worldwide, at concentrations ranging from sub-ng/L up to 27 mu g/L, as well as in various environmental aqueous matrices up to 13.7 mu g/L, highlighting its recalcitrance to conventional biological treatment. Nevertheless, the degradation potential of AMP via advanced chemical oxidation processes, which are widely considered as promising alternatives of conventional technologies, hardly has been investigated. Thus, this study aims at assessing the efficiency of solar photo-Fenton process at degrading AMP from secondary-treated wastewater effluents under various experimental conditions. The degradation rate of AMP via solar photo-Fenton oxidation fitted well the pseudo-first-order kinetic model. AMP was completely degraded after 30 min of treatment at various H2O2 doses (25-100 mg/L) under acidic pH, while its degradation was pronounced at mild alkaline conditions (inherent pH) (100% removal after 5 min). Total cultivable and AMP-resistant E. coli was completely inactivated after 180 min of treatment at the optimum operational conditions, and no regrowth was observed. Finally, the ecotoxicity results showed that the process was capable of eliminating the toxicity towards Daphnia magna after 180 min, providing thus a more complete evaluation of the overall efficiency of the process.