The emergence of multidrug resistance in bacterial pathogens has increased drastically and it has become prevalent in clinical infections. In last few decades, there is a large gap in the discovery of new antibiotics with novel mode of action. The situation of antimicrobial resistance has become so alarming that if not action is taken, infectious diseases will become major cause of global mortality and morbidity by 2050. The growing interest of researchers in nanotechnology and their possible application in healthcare is being seen as a new hope in discovery of novel antimicrobial agents. Among various approaches employed for the nanoparticle synthesis, biological methods are considered more advantageous and environment friendly. Biofilms are considered as novel target for the development of new antimicrobial entities. In this study, cerium oxide nanoparticles (CeO2 -NPs) were synthesized using Acorus calamus aqueous extract and tested for the antibiofilm activity both against Gram +ve and Gram -ve bacteria. The average size of synthesized CeO2 -NPs was found to be 22.03 nm. The biofilms of the test bacteria were inhibited by more than 75% by the treatment with CeO2 -NPs. The quantitative biofilm data were further verified by light microscopy, electron microscopy, and confocal microscopy. The confocal and electron microscopic analysis confirmed that treatment with CeO2-NPs reduced the development and colonization of the bacteria on solid support. Moreover, it was found that the colonization and biofilm development by test bacteria were fairly reduced on the glass surface. Moreover, a dose-dependent inhibition of preformed biofilms was also found. The exopolysaccharides (EPS) production by the test bacteria were substantially reduced by the supplementation of CeO2 -NPs in culture media. The findings of this study highlight the efficacy of cerium oxide nanoparticles against bacterial pathogens that may be exploited for the development of new alternative antimicrobial agent.