In the context of the expansion of antimicrobial resistance and the risk associated with nosocomial infections as a result of biofilm formation on surfaces and medical devices, this study presents the antibiofilm efficiency of zinc-based nanoparticles synthesized by an ecological method using aqueous extract of vine leaves (Vitis vinifera). Vine waste is a complex biological source and available in important quantities, given Romania’s viticultural potential. The antimicrobial properties of zinc oxide (ZnO) are well known, but the use of conventional ZnO has its limitations in terms of efficiency and biological compatibility. The sustainable synthesis of zinc nanoparticles offers not only an environmentally friendly solution, but also superior antimicrobial characteristics, suitable for advanced medical applications. Aqueous vine extract is a natural source of polyphenols, flavonoids, and other antioxidant substances necessary to reduce zinc ions. The zinc-based nanoparticles, obtained by green synthesis, were characterized by ultraviolet-visible (UV-vis) spectroscopy, Fourier transformation infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) coupled with energy dispersive X-ray spectroscopy (EDX). The antimicrobial potential of these zinc nanoparticles obtained by green synthesis has been evaluated against Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter baumannii, species that have the ability to form biofilm on medical surfaces and equipment. The working protocol included testing bacterial suspensions (500 μL) in combination with three concentrations (25, 50, and 100 μg/mL) of zinc nanoparticles. After incubation for 24 h, the biofilm was dispersed by vortexing and sonication, and viable cells were quantified by the colony-forming unit (CFU) counting method. Preliminary results indicate a significant inhibition of biofilm formation, dependent on concentration, especially in Gram-positive strains.