The fabrication of a new antibacterial system is an essential medical requirement to treat wounds caused by multidrug-resistant Gram-negative bacteria, mostly Pseudomonas aeruginosa. Antibacterial photodynamic therapy (APDT) is an encouraging therapy modality for bacterial infection. Anyhow, the applications of most photosensitizers are restricted by their poorwater solubility and their inefficiency in disruption of the outer membrane of Gram-negative bacteria. In this context, zinc phthalocyanine-colistin (ZnPc-Col) conjugate as a newphotosensitizerwas synthesized in the hope of improving interaction of phthalocyanine with the surface of Gram-negative bacteria. Overcoming the low solubility of phthalocyanine, was achieved by the incorporation of ZnPc-Col into chitosan hydrogel. The weight percentages of ZnPc-Col and glutaraldehyde had remarkable effects in determining the hydrogel microstructure. Different spectroscopic methods, SEM, and rheological measurements were used to assess the properties of ZnPc-Col and prepared hydrogels. The photosensitizing activity of the hydrogels was investigated by application of 1,3-diphenylisobenzofuran as a singlet oxygen chemical quencher. Hydrogels bearing zinc phthalocyanine-colistin adduct revealed an improved APDT efficiency compared to hydrogels containing just zinc phthalocyanine component. This enhancement has been seen against P. aeruginosa.