Zinc oxide (ZnO) nanoparticles (NPs) has become as promising candidate for antibacterial agents against Escherichia coli (E.coli), commensal hospital- acquired infections (HAIs). This study investigates the antibacterial action of ZnO NPs in three difference shapes; nanorod, nanoflakes and nanospheres against E.coli ATCC 25922. The antibacterial activity of ZnO NPs was determine through two standard protocols known as Clinical Laboratory Standards Institute (CLSI) MO2-A11 under light conditions of 5.70 w/m2 and American standard test method (ASTM) E-2149. Preliminary screening shows ZnO NPs did not inhibit the growth of E.coli. Further analysis using ASTM E-2149 in dynamic conditions revealed antibacterial activity after 3 hours with 100% reduction for ZnO NPs nanoflakes and 6 hours with 94.63% reduction for ZnO nanospheres, respectively. It demonstrated the ZnO NPs in nanoflakes and nanospheres exerted higher antibacterial activity possibly through release of ios, free radicals, ROS generation and electrostatic collision which contribute to bacterial death. Further analysis is needed to investigate biocompatibility of these samples for future biomedical applications.