Chemical pre-treatment of synthetic Procaine Penicillin G (PPG) effluent with ozone (applied dose = 1440 mg/h; treatment duration = 60 min) at pH = 7 was investigated. Successive biological treatability studies were performed with raw, ozonated penicillin formulation effluent and synthetic readily biodegradable substrate as simulated domestic wastewater. The PPG effluent additions were adjusted to constitute approximately 30% of the total COD in the reactor. Ozonation of PPG effluent resulted in practically complete removal of the parent pollutant accompanied by 40% COD abatement. Speaking for the raw PPG effluent, prolonged acclimation periods were necessary to obtain significant COD removal efficiencies. Batch activated sludge treatment experiments and respirometric studies have demonstrated that the selection of true retention time is extremely crucial for having high amount of slowly hydrolysable substrate or complex wastewater, like pharmaceutical effluent. The effect of ozonation time on biological treatability performance of PPG has been evaluated in the study. Pre-ozonation of PPG effluent did not improve its ultimate biodegradability.