The chitosan-copper oxide (Chi-CuO) biopolymer nanocomposites were synthesized by a simple green chemistry method using ascorbic acid as a reducing and capping agent. The intense peak around 300 nm was observed in the UV-visible spectrum indicating the formation of CuO nanoparticles. The prepared Chi-CuO nanocomposites were characterized using energy-dispersive X-ray spectroscopy (EDX), scanning electron microscopy (SEM), X-ray diffraction spectroscopy (XRD), and Fourier transform-infrared spectroscopy (FT-IR). SEM and XRD pattern showed cubic shape for Chi-CuO nanocomposites with average crystalline size of 17 nm, as calculated using Debye-Scherrer's formula. The FT-IR spectral studies showed the Cu-O bond formation with chitosan to form nanocomposites. Synthesized nanocomposites showed significant anti-microbial activity against Bacillus subtilis, Pseudomonas aeruginosa, Escherichia coli and Penicillium notatum, assayed using the agar well diffusion method. It also showed sporicidal activity against B. subtilis and exhibited effective biofilminhibitory activity against B. subtilis (69%/100 μg/mL) and P. aeruginosa (63%/100 μg/mL).