This study presents the green synthesis of zinc oxideâ��chitosan nanoparticles (Zin-Chi-NPs) using Sepia pharaonis cuttlebone as a marine biowaste source and chitosan as a natural stabilizing agent. X-ray diffraction (XRD) analysis confirmed a crystalline structure with 69.8 crystallinity, while Fourier-transform infrared spectroscopy (FTIR) revealed characteristic functional groups such as hydroxyl (3366 cmâ�»¹), amine (1566 cmâ�»¹), and carboxyl (1354 cmâ�»¹), indicating effective biogenic stabilization. Field emission scanning electron microscopy (FESEM) reveals densely packed, irregularly shaped nanoparticles dispersed across the chitosan matrix. Zin-Chi-NPs exhibited notable antibacterial activity, with inhibition zones of 15 ± 0.12 mm (E. coli), 18 ± 0.12 mm (Streptococcus mutans), and 19 ± 0.14 mm (Staphylococcus aureus); no inhibition was observed against Candida albicans. Cytotoxicity was evaluated on KB1 oral cancer cells using the MTT assay. A dose-dependent response was observed, with 18 cell death at 10 µg/mL and a marked increase to 97 at 200 µg/mL. The ICâ� â�� was calculated as 38.9 µg/mL, suggesting moderate cytotoxic efficiency and potential therapeutic relevance at higher concentrations. Overall, the synthesized Zin-Chi-NPs demonstrate promising structural stability, antimicrobial activity, and dose-dependent cytotoxicity, supporting their potential application in biomedical and pharmaceutical fields. © 2025 Elsevier B.V., All rights reserved.