Several families of photosensitizers are currently being scrutinized for antimicrobial photodynamic therapy applications. Differences in physical and photochemical properties can lead to different localization patterns as well as differences in singlet oxygen production and decay when the photosensitizers are taken up by bacterial cells. We have examined the production and fate of singlet oxygen in Escherichia coli upon photosensitization with three structurally-different cationic photosensitizers, namely New Methylene Blue N (NMB), a member of the phenothiazine family, ACS268, a hydrophobic porphyrin with a single cationic alkyl chain, and zinc(II)-tetramethyltetrapyridinoporphyrazinium salt, a phthalocyanine-like photosensitizer with four positive charges on the macrocycle core. The kinetics of singlet oxygen production and decay indicate different localization for the three photosensitizers, whereby NMB appears to localize in an aqueous-like microenvironment, whereas ACS268 localizes in an oxygen-shielded site, highly reactive towards singlet oxygen. The tetracationic zinc(II) tetrapyridinoporphyrazine is extensively aggregated in the bacteria and fails to produce any detectable singlet oxygen.