Active hybrid microspheres were prepared by blending aqueous solutions of gelatine and chitosan with different concentrations of copper nanoparticles (0) (CuNPs) using emulsion-based crosslinking synthesis to obtain hybrid microspheres. The incorporation of CuNPs slightly affected the physical and chemical properties of the films. Microscopic surface structure (SEM), energy dispersive X-ray (EDX) spectroscopy and X-ray diffraction (XRD) analysis confirmed the presence of elemental copper and the crystalline structure of the CuNPs in the hybrid matrix. The surface properties of CuNPs were studied by XPS analyses. The antimicrobial activity of CuNPs coated with chitosan (QO)/gelatine (GE) compared to the QO/GE matrix alone was investigated, using the agar diffusion and culture medium methods in Mueller-Hinton. The evaluation was performed using the Gram-negative bacterium E. coli and the Gram positive bacterium E. faecalis. The investigated microspheres showed antimicrobial activity. Hybrid microspheres with 40 mg of Cu, evaluated in liquid medium, inhibited the growth of E. coli by 56% and of E. faecalis by 40% compared to the matrix hybridised alone; in solid medium, the inhibition was 2.5-fold and 2.6-fold, respectively. Thus, these microspheres are a promising material for applications with medical uses.