Photodynamic treatment combining light and a photosensitizer molecule can be an effective method to inactivate pathogenic bacteria. This study identified vitamin K5 as an efficient photosensitizer for ultraviolet light A (UVA)-induced bacterial inactivation. Six bacterial species, Bacillus cereus (vegetative form), Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Klebsiella pneumoniae, and two species of antibiotic-resistant bacteria, Pseudomonas aeruginosa* and Staphylococcus aureus*, were suspended in aqueous solutions with or without vitamin K5 and exposed to UVA irradiation. UVA irradiation (5.8 J cm−2) with vitamin K5 (1600 μmol l−1) reduced the colony forming units (CFU) of these bacteria by three to seven logs. Antibiotic resistant bacteria were also susceptible to the bactericidal effects of UVA and vitamin K5 combination treatment. Inactivation of bacteria in human plasma required higher doses of UVA light and vitamin K5. UVA irradiation (30 J cm−2) with vitamin K5 (2000 μmol l−1) reduced E. coli and S. aureus spiked into human plasma by seven logs CFU/ml. Reactive oxygen species, such as superoxide anion radicals and hydroxyl radicals, were found to be generated in vitamin K5 aqueous solution after UVA irradiation, suggesting these oxygen species may mediate the inactivation of the bacteria.