Immobilization of photosensitizers in polymers opens prospects for their continuous and reusable application. Methylene blue (MB) and Rose Bengal were immobilized in polystyrene by mixing solutions of the photosensitizers in chloroform with a polymer solution, followed by air evaporation of the solvent. This procedure yielded 15–140 μm polymer films with a porous surface structure. The method chosen for immobilization ensured 99% enclosure of the photosensitizer in the polymer. The antimicrobial activity of the immobilized photosensitizers was tested against Gram-positive and Gram-negative bacteria. It was found that both immobilized photosensitizers exhibited high antimicrobial properties, and caused by a 1.5–3 log10 reduction in the bacterial concentrations to their total eradication. The bactericidal effect of the immobilized photosensitizers depended on the cell concentration and on the illumination conditions. Scanning electron microscopy was used to prove that immobilized photosensitizers excited by white light caused irreversible damage to microbial cells. Photosensitizers immobilized on a solid phase can be applied for continuous disinfection of wastewater bacteria.