AbstractDiketopyrrolopyrrole (DPP) derivatives containing sulfonamide (Sulfonamide‐DPP), pyridyl (Dipyridyl‐DPP) and N‐methylpyridyl (MePyridyl‐DPP) substituents were assessed as antibacterial photosensitizers. Non‐charged DPPs showed an intense absorption band centered at about 480 nm and green fluorescence emission (ΦF ~ 0.7) in acetonitrile. The absorption of MePyridyl‐DPP was bathochromically shifted at 510 nm, with decreased fluorescence emission. Sulfonamide‐DPP and Dipyridyl‐DPP photosensitized the formation of O2(1Δg) (ΦΔ ~ 0.15–0.17), while the production induced by MePyridyl‐DPP was at least 10 times lower. Furthermore, these DPPs produced a photoreduction of NBT similar to that of the control. Photodynamic inactivation induced by DPPs was first investigated at the single‐bacterium level of Staphylococcus aureus attached to a surface. After 30 min irradiation, MePyridyl‐DPP produced a complete eradication of the bacteria. In bacterial cell suspensions, dicationic DPP induced more than 7 log10 decrease in S. aureus cell survival after 30 min irradiation. Potentiation with iodide anions allowed a complete elimination of bacteria after 15 min therapy. This compound was also effective to eliminate S. aureus cells on biofilms. The results show that MePyridyl‐DPP bearing two positive groups provides an amphiphilic character to the structure that improves the interaction with the cell envelop. This effect enhances the photocytotoxic activity of MePyridyl‐DPP against bacteria.