Photodynamic therapy could provide an alternative to antibiotics for the treatment of local infections since a wide range of microorganisms have been shown to be susceptible to killing by photodynamic action (PDA) in vitro. The purpose of this study was to determine whether PDA was also able to affect the potency of two key bacterial virulence factors—lipopolysaccharide (LPS) and proteases. Suspensions of LPS from Escherichia coli and culture supernatants containing proteases of Pseudomonas aeruginosa were exposed to red light in the presence of toluidine blue O (TBO). The activity of each virulence factor was determined before and after irradiation. The limulus amoebocyte lysate (LAL) assay and the induction of proinflammatory cytokine (interleukin-8 and -6) release from human peripheral blood mononuclear cells (PBMC) were used for assessing the biological activity of LPS. Protease activity was quantified by azocasein hydrolysis. The biological activities of the LPS (both the LAL activity and its ability to induce cytokine release from PBMC) and the proteases were reduced significantly by irradiation with red light in the presence of TBO in a dose-dependent manner with respect to both the light energy dose and the TBO concentration. The ability of TBO-mediated PDA to reduce the activities of key virulence factors may be an additional benefit of using light-activated antimicrobial agents in the treatment of infectious diseases.