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Pulpotomies and pulpectomies are the most common clinical approach for dental caries in the primary dentition. Reinforced zinc oxide eugenol (ZOE) is an ideal material for filling in the pulp chamber after pulp therapies. The aim of this study was to assess the addition of Cloisite 5A nanoclay material to ZOE and evaluate its antibacterial properties. In this case–control study, the nanoclay nanoparticles were dissolved using a solvent (Eugenol) in different concentrations and their antibacterial properties were assessed using the agar diffusion test and biofilm analysis of <i>Streptococcus mutans</i> (<i>S. mutans</i>), <i>Enterococcus faecalis</i> (<i>E. faecalis</i>), and <i>Escherichia coli</i> (<i>E. coli</i>) in in vitro conditions using the AATCC 100 standards. The diameter of the inhibition zone was measured and assessed statistically using the SPSS software (Version 28, IBM, Chicago, IL, USA) with a significance level of 0.05. The antibacterial properties of the ZOE with nanoclay particles were significantly greater in comparison to the plain ZOE against <i>E. faecalis</i>, <i>S. mutans</i>, and <i>E. coli</i>. The inhibition zone against <i>E. coli</i> under the effect of the ZOE and nanoclay particles combined was significantly higher than that against <i>E. faecalis</i> and <i>S. mutans</i>. The current study showed that the addition of Cloisite 5A nanoclay particles can improve the antibacterial properties of ZOE significantly at certain concentrations.