The introduction of novel bacterial strains and the development of microbial approaches for nanoparticles biosynthesis could minimize the negative environmental impact and eliminate the concern and challenges of the available approaches. In this study, a biological method based on microbial cell-free extract was used for biosynthesis of ZnO NPs using two new aquatic bacteria, Marinobacter sp. 2C8 and Vibrio sp. VLA. The synthesized ZnO NPs were characterized by UV–Visible spectroscopy, Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), transmission electron microscopy (TEM), atomic force microscope (AFM), dynamic light scattering (DLS) and zeta potential. The UV–Visible absorption peak was found to be at 266 and 250 nm for ZnO-2C8 NPs and ZnO-VLA NPs, respectively. FTIR study suggested that the hydroxyl, amine, and carboxyl groups of bacterial proteins are mainly responsible for stabilizing the biosynthesized ZnO NPs. The formation of hexagonal wurtzite structure of ZnO NPs was confirmed by the XRD pattern. The morphology of the nanoparticles was found to be spherical with the average particle size of about 10.23 ± 2.48 nm and 20.26 ± 4.44 nm for ZnO-2C8 NPs and ZnO-VLA NPs, respectively. The values of zeta potential indicate the high stability of the biosynthesized ZnO NP. Zeta potential values indicated the high stability of the biosynthesized ZnO NP and were obtained −20.54 ± 7.15 and –23.87 ± 2.29 mV for ZnO-2C8 NPs and ZnO-VLA NPs, respectively. The biosynthesized ZnO NPs had antibacterial activity against Gram-negative and Gram-positive strains and possessed excellent antibiofilm activity with the maximum inhibition of about 96.55% at 250 µg/mL. The DPPH activity of ZnO-2C8 NPs and ZnO-VLA NPs were found 88.9% and 85.7% for 2500 μg/mL concentration, respectively. The toxicity test revealed the biocompatibility of the biosynthesized ZnO NPs. The results suggested that this approach is a very good route for synthesizing ZnO NPs with potential applications in biotechnology.