In this present study, a hybrid Chi-Fe3O4 was prepared, characterised and evaluated for its antibacterial and antibiofilm potential against Staphylococcus aureus and Staphylococcus marcescens bacterial pathogens. Intense peak around 260 nm in the ultraviolet-visible spectrum specify the formation of magnetite nanoparticles. Spherical-shaped particles with less agglomeration and particle size distribution of 3.78-46.40 nm were observed using transmission electron microscopy analysis and strong interaction of chitosan with the surface of magnetite nanoparticles was studied using field emission scanning microscopy (FESEM). X-ray diffraction analysis exhibited the polycrystalline and spinel structure configuration of the nanocomposite. Presence of Fe and O, C and Cl elements were confirmed using energy dispersive X-ray microanalysis. Fourier transform infrared spectroscopic analysis showed the reduction and formation of Chi-Fe3O4 nanocomposite. The antibacterial activity by deformation of the bacterial cell walls on treatment with Chi-Fe3O4 nanocomposite and its interaction was visualised using FESEM and the antibiofilm activity was determined using antibiofilm assay. In conclusion, this present study shows the green synthesis of Chi-Fe3O4 nanocomposite and evaluation of its antibacterial and antibiofilm potential, proving its significance in medical and biological applications.