The aim of the present study was to synthesize CeO₂ nanoparticles using plant pathogenic fungus F. solani and also to study the antibacterial activity as well as the influence on the inhibition of biofilm formation against biomedically important bacterial strains namely Staphylococcus aureus, Psedomonas aeriginosa, Escherichia coli and Klebsiella pneumoniae. Thermogravimetric/differential thermal analysis (TG/DTA) suggested a crystallization temperature of the as-synthesized CeO₂ nanopowder at 400 °C. Powder X-ray diffraction analysis and Raman spectroscopy substantiated the presence of CeO₂ nanoparticles with a cubic fluorite structure. The contribution of functional groups corresponding to the F. solani fungal supernatant for the synthesis of CeO₂ nanoparticles was studied by Fourier transform infrared (FTIR) spectroscopy. The room temperature photoluminescence spectrum of calcined CeO₂ nanopowder was recorded. Field emission scanning electron microscopy (FESEM) equipped with energy dispersive X-ray spectroscopy (EDAX) ascertained the formation of homogeneously distributed spherically shaped CeO₂ nanoparticles. Furthermore, transmission electron microscopy (TEM) demonstrated the spherical morphology of the CeO₂ nanoparticles having sizes ranging from 20 to 30 nm and also the selected area electron diffraction (SAED) pattern revealed the polycrystalline nature of the CeO₂ nanoparticles, which is consistent with the XRD results. The presence of surface oxidation states Ce (3d) and O (1s) of the CeO₂ nanoparticles was confirmed by X-ray Phoelectron Spectroscopy (XPS) analysis. The antibacterial activity of CeO₂ nanoparticles was evaluated by the disc diffusion method and it showed the highest activity against P. aeruginosa as well as K. pneumoniae. In addition, the inhibition on biofilm formation by CeO₂ nanoparticles has also been examined by confocal laser scanning microscopy (CLSM). Furthermore, the electrochemical property of the biosynthesized CeO₂ nanoparticles was studied by the cyclic voltammetry technique.