Bacterial infections remain a critical global health concern, often leading to prolonged self-medication, extended healing times, treatment failures, increased hospitalization, and higher healthcare costs, alongside increased mortality rates. Recent research efforts have focused on enhancing human health and environmental sustainability through innovative synthesis techniques. This study presents a straightforward and cost-effective method for the green synthesis of zinc oxide nanoparticles (ZnO NPs) using fruit extract from Washingtonia robusta H. Wendl and investigated their antibacterial activity. The W. robusta fruit extract underwent high-performance liquid chromatography (HPLC), resulting in the isolation of four distinct fractions that were tested for antibacterial activity against various Gram-positive and Gram-negative bacteria. The methanolic fraction demonstrated the highest antibacterial potential and was analyzed for bioactive compounds using gas chromatography-mass spectrometry. This analysis identified ten biochemical constituents, including hexasiloxane (43.28%), E-9-tetradecenoic acid (12.99%), oleic acid (10.12%), and heptasiloxane (9.83%). The ZnO NPs using methanolic fraction and characterized using various techniques, including ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, zeta potential analysis, and transmission electron microscopy. The antibacterial efficacy of the ZnO NPs was evaluated against Escherichia coli ATCC25922, and Bacillus cereus ATCC6633. The antibacterial activity was assessed. Results indicated that the ZnO NPs exhibited a good antibacterial activity with average inhibition zones 18 mm against E. coli, and 24 mm against B. cereus. The MIC values for ZnO NPs were 35 µg/ml against E. coli and 25 µg/ml against B. cereus. The MBC values matched with MIC results that indicated the potent bactericidal action of the prepared ZnO NPs.