The aim of the study is to biosynthesize and characterize Zinc oxide nanoparticles (ZnO-NPs) and to evaluate their antibacterial potential. Several isolates of the actinomycetes strain were isolated from marine sediments, and the strain was morphologically identified as actinomycetes and screened for ZnO-NPs synthesis. N5 strain showed potent activity while screening against human pathogens and further ZnO-NPs were synthesized for anti-bacterial studies. The ZnO-NPs were characterized using physio-chemical techniques such as ultraviolet spectroscopy, Fourier- Transform Infrared spectroscopy (FTIR), and X-Ray diffraction (XRD). The morphology of ZnO-NPs was analyzed using Scanning Electron Microscopy (SEM)and revealed cubic shaped. The synthesized ZnO-NPs were subjected for their antibacterial potential against human pathogens of both gram-positive and negative bacteria, using the agar well diffusion method. The ZnO-NPs showed positive results on both pathogens, Staphylococcus showed higher activity, followed by E. coli. The synthesized ZnO-NPs have good antibacterial activity which pay way to develope new antimicrobial compounds in pharmaceutical sectors.