In this study biopolymer-inorganic material of chitosan‑copper oxide-neem seed (CS-CuO-NS) biocomposite was successfully synthesized by simple precipitation method and characterized by FT-IR, XRD, HR-SEM, TEM and TGA analyses. From HR-SEM and TEM analysis, CS-CuO-NS biocomposite shows flower and needle like structure respectively. The size of the as prepared CS-CuO-NS biocomposite is found to be 20-100 nm. All the synthesized materials were tested for antibacterial activity against both gram positive like Staphylococcus aureus (S. aureus) and Streptococcus pyogenes (S. pyogenes) and gram negative like Escherichia coli (E. coli) and Klebsiella aerogenes (K. aerogenes) bacterial strains. The maximum zone of inhibition is obtained for CS-CuO-NS biocomposite against S. aureus (23 mm), S. pyogenes (21 mm), E. coli (22 mm) and K. aerogenes (20 mm). The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined. The antioxidant activity was determined by free radicals scavenging such as 1, 1-Diphenyl-2-picryhydrazyl (DPPH) and 2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS). Furthermore, the cytotoxicity effect was investigated against human breast cancer (MCF-7) cell line and the highest cytotoxicity (IC₅₀:16.33 μg/mL) is found to be in biocomposite. From the results of antibacterial, antioxidant and cytotoxic activities, it is concluded that CS-CuO-NS biocomposite may be suitable for biomedical applications.