Antibiotic pollution is one of the factors contributing to the spread of antibiotic-resistant bacteria in the environment. Advanced oxidation and irradiation processes have been introduced to eliminate antibiotics from water and wastewater. However, few studies have reported the toxic effects of residual antibiotics and their byproducts induced by a treatment system. In this study, we compared the efficacies of chemical (high-performance liquid chromatography [HPLC]) and biological (antimicrobial susceptibility test) assays for measuring the concentrations of residual antibiotics after gamma irradiation for degrading amoxicillin, cephradine, lincomycin, and tetracycline. The concentrations of residual antibiotics estimated using the two assay methods were almost identical, except cephradine. In the case of cephradine, inhibited bacterial growth was observed that was equivalent to twice the concentration measured by HPLC in the samples subjected to gamma irradiation. The observed inhibition of bacterial growth suggested the generation of potentially toxic intermediates following antibiotic degradation. These results indicate that biological and chemical assays should be used in concert for monitoring antibiotic contamination and the toxic derivatives of antibiotic degradation. The results demonstrate that these four antibiotics can be decomposed by 2.0 kGy gamma-irradiation without toxic effects of their byproducts.