ABSTRACT Objective: The present investigation looked into PLGA micron-sized particles or NPs, loaded with calcium hydroxide for canal disinfecting processes which have antimicrobial properties, given the significant rejection probabilities related to these kinds of treatments. Methodology: The MIC and inhibitory growing zones (n = 5) were found using the broth dilution and agar diffusion tests, accordingly. Employing confocal microscopy and live/dead labelling, cell viability was evaluated (n = 5). ANOVA was used to evaluate, and post hoc analysis came next. Results: Ca(OH)2 NPs showed a minimum inhibitory concentration of 5 μg/mL for Fn and 10 μg/mL for Pg and Ef (P < 0.001), following a 24-hour incubation period. Conclusion: To sum up, Ca(OH)2-contained PLGA NPs show encouraging ability to kill microbes during canal cleaning treatments.