The aim of this study was to evaluate the antimicrobial activity of a new experimental nano-hydroxyapatite epoxy resin-based sealer (Nanoseal) with several other commercially available sealers; AH26, Tubliseal, Sealapex and Roekoseal against Enterococcus faecalis, Pseudomonas aeruginosa, Streptococcus mutans, Streptococcus sobrinus and Escherichia coli for up to 7 days. Agar diffusion was used in this study. Fifty Muller-Hinton agar plates were prepared and divided into five experimental groups (n = 10), for each micro-organism. Another 10 agar plates were used as positive and negative controls. Endodontic sealers were tested against each micro-organism. Inhibition zones produced were recorded. The results of this study showed that all test materials exhibited inhibition zones towards the tested micro-organisms for 7 days except for Roekoseal, which showed no inhibition zones. Nanoseal and AH26 exhibited similar zones of inhibition. Significant difference was found between Nanoseal and the other tested sealers (P < 0.001).