Advanced oxidation processes (AOPs) composed of O3 and UV were applied to degrade penicillin (PEN). The degradation efficiency was evaluated in terms of changes in the absorbance (ABS) and total organic carbon (TOC). The combination of O3/H2O2/UV and O3/UV showed the best performance for the reduction of ABS (100% for 9 min) and TOC (70% for 60 min) values, although the mineralization was uncompleted under the experimental condition in this study. The change in biotoxicy was monitored with Escherichia coli susceptibility and Vibrio fischeri biofluorescence. The E. coli susceptibility was eliminated completely for 9 min by O3/UV, and the toxicity to V. fischeri biofluorescence was 57% reduced by O3/H2O2/UV. For the ultimate treatment of PEN, it is suggested that an AOP using O3/UV is followed by biological treatment, utilizing the enhanced biodegradability by the AOP. During 30 min of O3/UV treatment, the BOD5/COD ratio as an indication of biodegradability showed about 4-fold increment, compared to that of using a non-treated sample. TOC removal rate for AOP-pretreated PEN wastewater increased 55% compared to that of using the non-pretreated one through an aerobic biological treatment by Pseudomonas putida for artificial wastewater containing 20 mg/L of PEN. In conclusion, O3/UV process is recommended as a pretreatment step prior to an aerobic biological process to improve the ultimate degradation of penicillin.