Bacteria can form biofilms on any surface, which causes biofilm-associated infections and bacterial resistance to antibiotics. Thus, it is important to design new-generation non-chemotherapeutic nanoagents for effective antibacterial and antibiofilm strategies. Herein, the effects of the anchoring groups, which are imidazole and carboxylic acid, of zinc phthalocyanines (ZnPcs) sensitized TiO2 on Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) were investigated under light-emitting diode (LED) irradiation. The photocatalytic antibacterial activities of ZnPc-1/TiO2 and ZnPc-2/TiO2 on the bacterial strains were examined by monitoring the optical density value at 600 nm (OD600 nm). Glutathione (GSH) oxidation assay was used to measure the reactive oxygen species (ROS) generation activity of the compounds. Bacterial damages were imaged by scanning electron microscopy (SEM). According to our photocatalytic antibacterial mechanism, photogenerated electrons are transferred from Pcs to TiO2 and then react with O2, thus creating ROS, which causes damage to bacterial membrane, protein and biofilm destruction as well. Further, computational simulation analysis was used to show the interaction patterns of ZnPc-1 and ZnPc-2 with penicillin binding protein 2a (PBP2a) of S. aureus and FimH lectin protein (PDB:4XO8) of E. coli to elucidate the dark molecular antibacterial mechanism of the compounds. The obtained results from computational studies showed that ZnPc-2 binds firmly through bonds with the 1MWT protein from S. aureus. On the other hand, ZnPc-1 binds firmly through bonds with the 4XO8 protein from E. coli. From combining experimental and computational results, we can conclude that this strategy can be applied to different types of bacterial infections.