Nosocomial infections are a dangerous complication of various surgical procedures or injuries because the pathogens causing them are resistant to antibiotic therapy. Antibacterial photodynamic therapy (aPDT), which is based on  the suppression of the growth of pathogenic microorganisms by means of free radicals and reactive oxygen species generated by the irradiation of substances with photosensitising activity, can be an effective method for the  treatment of nosocomial infections. In this work, we have shown that the endogenous photosensitiser riboflavin, which has low dark toxicity, good water solubility and high quantum yield, can be used as an agent for aPDT. To this end,  we investigated the antibacterial activity of riboflavin against E.Coli bacteria transformed with the mKate protein using blue and near-ultraviolet light sources in different irradiation modes. It was shown that half-maximal inhibition of bacterial growth could be achieved at concentrations of 0.1–0.5 mg/ml riboflavin when irradiated at a wavelength of 365 nm and at 0.6–1.2 mg/ml when irradiated at a wavelength of 450 nm, 10–20 minutes of irradiation. The  proposed approach may be promising for the treatment of nosocomial infections, including those resistant to antibiotics.