Aim This study was performed to investigate the possibility to photodynamically inactivate Gram-negative bacteria without intracellular uptake of the photosensitizer. The efficiency of the photodynamic growth inhibition of Escherichia coli (SURE2) was proved in a comparative study of a neutral and a cationic photosensitizer. Methods and Results We used confocal laser scanning microscopy (CLSM) to investigate the uptake of the photosensitizer by the bacteria to show that both chlorin e6 and TMPyP are not accumulated in the cells. Fluorescence lifetime imaging (FLIM) and phototoxicity experiments were used to investigate the photodynamic inactivation of the Gram-negative bacteria. The phototoxicity experiments were carried out using a white light LED-setup to irradiate the bacterial suspensions. The viability of the bacteria was obtained by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-assay. For the cationic TMPyP, photodynamic inactivation without intracellular uptake was observed, whereas for chlorin e6 such behaviour was not found. Conclusions It was proven that in general, it is possible to photodynamically inactivate Gram-negative bacteria without photosensitizer accumulation in the bacterial cells. This fact is especially interesting, considering that the development of resistances may be prevented, leaving the active components outside the bacterium. Significance and Impact of the Study In a world with bacteria that gain the ability to withstand the effects of antibiotics and are able to transmit on these resistances to the next generation, it becomes necessary to develop new approaches to inhibit the growth of multi-resistant bacteria. The photodynamic inactivation of bacteria is based on a three-component system by which the growth of the bacterial cells is inhibited. The well-directed oxidative damage is initiated by visible light of a certain wavelength, which excites a nontoxic photoactive molecule, called photosensitizer. Its reaction with oxygen causes the generation of cytotoxic species like singlet oxygen, which is highly reactive and causes the inactivation of the growth of bacteria.