Present study deals with the green fabrication of copper oxide nanoparticles (CuO NPs) employing cell-free aqueous extract of Cladophora glomerata (L.) Kuetz, freshwater algal species. The UV-visible, FTIR, XRD, FESEM, HRTEM, EDX, BET, XPS and Raman spectroscopic techniques were used to confirm and characterize the biosynthesized CuO NPs. The UV-Vis analysis revealed a sharp peak at 264 nm with a band gap of 3.7 eV, which was attributable to the fabrication of CuO NPs. FESEM and HRTEM detect the spherical-shaped morphology with size between 40 and 50 nm. The biochemical profiling of cell free extract of the C. glomerata by Gas chromatography-mass spectrometry (GC-MS) revealed the presence of various bioactive biomolecules that may acts as a precursor for the fabrication of CuO NPs. The antibacterial study of fabricated CuO NPs revealed significant growth inhibitory potential against selected bacterial strains Klebsiella pneumoniae and Bacillus cereus with an IC