The photodynamic inactivation by near UV and α-terthienyl (αT) of bacteria immobilized in poly(methyl methacrylate) (PMMA) was observed. Killing curves for Escherichia coli strains exhibited multi-hit kinetics, whereas curves for Pseudomonas aeruginosa and Staphylococcus aureus exhibited single-hit kinetics. Cell inactivation by irradiated αT film at an immobilized concentration of 20 μg/cm2 was substantially caused by generated singlet oxygen (1O2), but not by that from free αT molecules released from the film after irradiation with near UV. The inactivation rate was found to depend on the surface density of αT in the film. A linear relationship between the concentration of 1O2 generated on the film surface and the apparent rate of cell death was obtained.