Microwave assisted synthesis of ZnO nanoparticles was performed at varying pH of the synthesis medium. At pH 8 and 10, uniform and crystalline ZnO nanoparticles were formed whereas at pH 6, layered basic zinc acetate (LBZA) was obtained. The present work proposes a new strategy to synthesize LBZA via microwave irradiation method. As-prepared ZnO nanoparticles were used to study antibacterial behavior against pathogenic Gram-negative and Gram-positive bacteria viz. E. coli, P. aeruginosa, S. aureus and E. faecalis under dark condition. The results show a significant decrease in viability of pathogenic bacterial cells with increase in interaction time with ZnO nanoparticles.