Nowadays, with the growing need for alternative antibacterial materials for the treatment of bacterial infections, TiO2 with antibacterial properties has attracted attention as a potential antibacterial agent. Ni-TiO2 and Co-TiO2 nanofibers (NFs) were synthesized via an electrospinning process. The antibacterial activities of these NFs against S. aureus and E. coli were evaluated under UV-light illumination using optical density measurements. Co-TiO2 exhibited superior antibacterial activity against both S. aureus and E. coli under UV-light irradiation. The antibacterial mechanism was further investigated through a glutathione (GSH) oxidation assay and morphological analysis using scanning electron microscopy (SEM). Hydrophilicity was evaluated by contact angle measurement. The antibiofilm activities of TiO2, Ni-TiO2, and Co-TiO2 NFs were investigated with respect to E. coli and S. aureus biofilms. Ni-TiO2 and Co-TiO2 demonstrated more effective antibiofilm activities than bare TiO2. Under UV-light irradiation, the biofilm inhibition efficacy was increased for both Ni-TiO2 and Co-TiO2 NFs while Co-TiO2 NFs were found to have the greater antibiofilm performance. Additionally, in silico analysis was conducted to explore the molecular interactions of the NFs with S. aureus Immunoglobulin-Binding B Domain (PDB ID: 1BDD) and FimH lectin protein of E. coli (PDB ID: 4XO8). Co-TiO2 exhibited stronger binding to S. aureus, while TiO2 showed stronger binding to E. coli.