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Chitosan was used in the sol-gel synthesis of zinc oxide nanoparticles (ZnO NPs) as a capping agent in order to control the size, morphology, optical bandgap, photocatalytic efficiency, and antimicrobial activity. Different chitosan sources were used for the sol-gel synthesis of ZnO NPs, namely chitosan of shrimp shells, crab shells, and Streptomyces griseus bacteria. The photocatalytic efficiency was studied by using the methylene blue (MB) photodegradation test, and the antibacterial activity of the different types of ZnO NPs was investigated by the agar well diffusion technique. The particle size of ZnO NPs varied between 20 and 80 nm, and the band gap energy ranged between 2.7 and 3.2 eV. Due to the different chitosan sources, the ZnO NPs showed different antibacterial activity against Listeria innocua, Bacillus Subtiliis, Staphylococcus Aureus, Salmonella Typhimurium and Pseudomonas Aeruginosa. The ZnO NPs with lower band gap values showed better antibacterial results compared to ZnO NPs with higher band gap values. The MB dye removal of ZnO (shrimp shells), ZnO (crab shells), and ZnO (Streptomyces griseus) reached 60%, 56%, and 44%, respectively, at a contact time of 60 min, a low initial MB dye concentration of 6 × 10−5 M, a solution temperature of 25 °C, and a pH = 7.