The present study reports the green synthesis of zinc oxide nanoparticles using the aqueous leaf extract of Laurus nobilis (Ln-ZnO NPs) by co-precipitation method. The synthesized Ln-ZnO NPs were characterized by UV–Vis spectroscopy, FTIR, XRD, TEM, SEM and EDX. Ln-ZnO NPs were crystalline in nature, flower like and have hexagonal wurtzite structure with a mean particle size of 47.27nm. The antibacterial activity of Ln-ZnO NPs was greater against Gram positive (Staphylococcus aureus) bacteria than Gram negative (Pseudomonas aeruginosa) bacteria. The zone of inhibition against S. aureus was 11.4, 12.6 and 14.2mm at 25, 50 and 75μgmL⁻¹. The zone of inhibition against P. aeruginosa was 9.8, 10.2 and 11.3mm at 25, 50 and 75μgmL⁻¹. The light and confocal laser scanning microscopic images evidenced that Ln-ZnO NPs effectively inhibited the biofilm growth of S. aureus and P. aeruginosa at 75μgmL⁻¹. The cytotoxicity studies revealed that Ln-ZnO NPs showed no effect on normal murine RAW264.7 macrophage cells. On the other hand, Ln-ZnO NPs were effective in inhibiting the viability of human A549 lung cancer cells at higher concentrations of 80μgmL⁻¹. The morphological changes in the Ln-ZnO NPs treated A549 lung cancer cells were observed under phase contrast microscope.