This research was performed to assess the elimination of antibiotics and the consequential loss of antibacterial activity in water using UV irradiation. The target compounds were amoxicillin (AM), ampicillin (AP), cephradine (CP), and penicillin G (PG), which belong to the β-lactam class of antibiotics. Of the four antibiotics examined, cephradine possessed the greatest photolysis rate compared with the other three antibiotics. The quantum yield (φM) and molar adsorption coefficient (ε254) at 254 nm were determined with the direct photolysis kinetic model. The photo-product formation of the four antibiotics was observed by scanning and comparing UV spectra (200 < λ > 400 nm) of the aqueous solutions before and after UV photolysis. In addition, biological assay was performed to investigate the antibacterial activity of both the original substances and their degradation products, which accrue during UV irradiation. In the cases of amoxicillin and ampicillin, the antibacterial potential of their photo-products was observed at the concentrations examined. Consequently, occurrence of photo-byproducts of the two antibiotics necessitates detailed study on the potential risk to the actual environment.