The light-induced antibacterial activity of symmetric and asymmetric oligophenylene ethynylenes (OPEs) was investigated against Gram-positive (Staphylococcus aureus and Staphylococcus epidermidis) and Gram-negative (Escherichia coli) bacteria. To understand the light-induced biocidal effect better, the transient absorption and triplet lifetime of OPEs were studied in methanol and water. A higher triplet lifetime was observed for OPE; samples in water than in methanol. The magnitudes of the changes in optical density (Delta OD) of the S-OPE-n(H) series of symmetric oligomers are much higher than that of the asymmetric OPE-n series in water and are generally correlated with the singlet oxygen yield. It was found that the antibacterial activity against both Gram-positive and Gram-negative bacteria is size-, concentration-, and time-dependent. The light-induced antibacterial activity may result from the coordinated interactions of membrane disruption and interfacial or intracellular singlet oxygen generation, and the dominant factor is most likely the latter. The results obtained in this study will aid in the design of more efficient biocides in the future.