AbstractThe development of novel drug candidates for treating microbial infections and cancer turns into a challenging task. The biopolymer‐based nanomaterials have gained popularity for their effective roles in the pharmaceutical sector. The present investigation aims to valorize crab shell wastes to extract chitosan. The obtained (chitosan) biomass was utilized to synthesize chitosan‐zinc oxide nanoparticles (Ch‐ZnO‐NPs). The nanomaterial characterization was achieved by a variety of physicochemical techniques including UV, Fourier transform infrared spectroscopy (FT‐IR), X‐ray diffraction (XRD), elemental analysis, and High‐resolution transmission electron microscopic (HR‐TEM) examination. The synthesized nanomaterials have a particle size range of 35–80 nm and are more stable with a zeta potential value of (−40.78) mv. Additionally, the designed nanomaterials were utilized to evaluate the biological efficiency. The minimal inhibitory concentration value of Ch‐ZnO‐NPs was observed as 10 μg/mL. The designed nanomaterials significantly reduced the growth of Vibrio alginolyticus and Bacillus cereus and inhibited biofilm formation. The synthesized Ch‐ZnO‐NPs unveiled cytotoxic effects on A549 cells with an IC₅₀ (half‐maximal inhibitory concentration) of 20 μg/mL. Furthermore, substantial increase in cell death was noticed in A549 cells treated with Ch‐ZnO‐NPs. Consequently, this investigation concludes that Ch‐ZnO‐NPs may act as a biomedicine.