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Global emergence and persistence of the multidrug-resistant microbes have created a new problem for management of diseases associated with infections. The development of antimicrobial resistance is mainly due to the sub-judicious and unprescribed used of antimicrobials both in healthcare and the environment. Biofilms are important due to their role in microbial infections and hence are considered a novel target in discovery of new antibacterial or antibiofilm agents. In this article, zinc oxide nanoparticles (ZnO-NPs) were prepared using extract of <i>Plumbago zeylanica</i>. ZnO-NPs were characterized and then their antibiofilm activity was tested against Gram-positive and Gram-negative bacteria. The ZnO-NPs were polydispersed, and the average size was obtained as 24.62 nm. The presence of many functional groups indicated that phytocompounds of <i>P. zeylanica</i> were responsible for the synthesis, capping, and stabilization of ZnO-NPs. Synthesized NPs inhibited the biofilm formation of <i>E. coli</i>, <i>S. aureus</i>, and <i>P. aeruginosa</i> by 62.80%, 71.57%, and 77.69%, respectively. Likewise, concentration-dependent inhibition of the EPS production was recorded in all test bacteria. Microscopic examination of the biofilms revealed that ZnO-NPs reduced the bacterial colonization on solid support and altered the architecture of the biofilms. ZnO-NPs also remarkably eradicated the preformed biofilms of the test bacteria up to 52.69%, 59.79%, and 67.22% recorded for <i>E. coli</i>, <i>S. aureus</i>, <i>P. aeruginosa</i>, respectively. The findings reveal the ability of green synthesized zinc oxide nanoparticles to inhibit, as well as eradicate, the biofilms of Gram-positive and Gram-negative bacteria.