Solvothermal synthesis was used to investigate the formation of zinc oxide (ZnO) nanoparticles (NPs). A series of ZnO NPs was synthesized with different relative ratios of didodecyldimethylammonium bromide (DDAB) and zinc nitrate (ZN). The variation in the molarity influenced the crystallinity, size, and morphology of the obtained ZnO NPs. X-ray diffraction, Fourier-transform infrared spectroscopy, field-emission scanning electron microscopy, high-resolution transmission electron microscopy, and zeta potential analysis were used to study the characteristic features of the ZnO NPs. The ZnO surface charge, size, and morphological structure were highly reliant on the concentrations of DDAB and ZN. With increasing relative ratio of DDAB to ZN, the particle size of ZnO NPs decreased and the surface charge increased to higher positive value. The ZnO NPs synthesized with cationic liquid DDAB presented enhanced performance in preventing the growth of Staphylococcus aureus (S. aureus) and Escherichia coli (E. coli) strains. The antibacterial activity of ZnO NPs have direct contact with the microbial cell wall resulting in destruction of bacterial cell integrity, release of antimicrobial Zn ions, and induce cell death. This is due to the positively charged smaller ZnO NPs, prepared with DDAB cationic surfactant, effectively acting as an antimicrobial agent against food-borne pathogenic bacteria.