Pyocyanin (PyO⁻) (1-hydroxy-5-methylphenazine) is a cytotoxic compound secreted by Pseudomonas aeruginosa, an omnipresent bacterium and a human pathogen. We report that visible light illumination in the presence of rose bengal, or riboflavin, in aerated solutions (pH 7.0–7.2) induces irreversible loss of the pigment's characteristic absorption band at 690 nm, indicating its oxidation. This photobleaching was paralleled by generation of a multiline Electron Paramagnetic Resonance (EPR) spectrum attributed to a PyO⁻-derived radical. The reaction was dependent on the presence of air, sensitizers and light, was inhibited by sodium azide and was unaffected by ethanol. This suggests that PyO⁻ was oxidized largely via singlet oxygen and that hydroxyl radicals were not involved. The photochemically modified pigment was less efficient in oxidizing NAD(P)H and generated less superoxide (by ∼50%) than the intact PyO⁻, indicating its partial inactivation. 1-Methoxy-5-methylphenazine, a PyO⁻ analog in which the –O⁻ moiety was replaced by the methoxy group (–OMe), was resistant to oxidation, suggesting that oxidation of PyO⁻ involves its phenolate moiety. These results also suggest that photosensitization could be a potentially useful method for inactivation of PyO⁻ and, possibly, detoxification of superficial wounds (skin, eye) infected with P. aeruginosa.