Zinc oxide nanoparticles (ZnONPs) have attracted considerable attention as antibacterial agents due to their superior physicochemical properties. This study aimed to synthesize ZnONPs via a green synthesis approach using Illicium verum flower extract, which is rich in phytochemical compounds, as a natural bioreductant, as well as to determine the optimal synthesis conditions and characterize the resulting nanoparticles. The synthesis was carried out using zinc acetate dihydrate, with variations in the precursor-to-extract volume ratio (1:1–1:4) and pH (7–11). Characterization was performed using UV–Vis spectrophotometry, Fourier Transform Infrared Spectroscopy (FTIR), and Particle Size Analysis (PSA), while antibacterial activity against Escherichia coli was evaluated using the disk diffusion method. The results showed that the optimal conditions were achieved at a volume ratio of 1:2 and a pH of 10, with a maximum absorption wavelength of 385 nm. The synthesized ZnONPs exhibited an average particle size of 18.27 nm with a low polydispersity index (0.1769), and the presence of Zn–O functional groups was confirmed by FTIR analysis. Furthermore, ZnONPs demonstrated very strong antibacterial activity against Escherichia coli, with an inhibition zone diameter of 32.49 mm, which was higher than that of the Illicium verum extract. These findings indicate that Illicium verum extract is an effective natural bioreductant for the green synthesis of ZnONPs and highlight the potential of the synthesized nanoparticles as efficient and environmentally friendly antibacterial agents. This study also demonstrates the feasibility of utilizing Illicium verum flower extract for ZnONP synthesis and antibacterial applications against Escherichia coli.