Intracanal medicaments are an important adjunct to the effective disinfection of the root canal system. However, conventional intracanal medicaments do not provide adequate protection against Enterococcus faecalis, which is the organism of interest in many cases of root canal failures. This study aimed to evaluate the influence of biosynthesized calcium oxide nanoparticles (CaO NPs) on the antibacterial activity, pH, microleakage and cytotoxicity of intracanal medicaments. CaO NPs were biosynthesized by the direct thermal decomposition of eggshells (EGS) and the reduction of calcium nitrate with papaya leaf extract (PLE). These nanoparticles were mixed with a proprietary calcium hydroxide powder in 10% and 25% (w/w) concentrations and blended in analytical-grade coconut oil to formulate the experimental medicaments. These were then evaluated for antibacterial activity, pH, microleakage and cytotoxicity at 1 day, 7 days and 15 days. A proprietary calcium hydroxide paste formulation (MX) was used as the control. Means and standard deviations were calculated and analyzed using repeated-measures ANOVA for pH and three-way ANOVA for the antibacterial effect, microleakage and cytotoxicity, followed by LSD post hoc analysis. Significant antibacterial activity was noted against Enterococcus faecalis at all times, with zones of inhibition (ZOI) up to 19.60 ± 2.30 mm. pH levels up to 13.13 ± 0.35 were observed for the experimental groups. Microleakage remained comparable to the control, while cytotoxicity was not observed in any of the groups at any time. Intracanal medicaments formulated with 10% and 25% (w/w) of biosynthesized CaO NPs could be promising candidates for the disinfection of the root canal system compared to conventional counterparts.