<h3>Background</h3> Endodontic sealers are continuously being improved to enhance performance after root canal treatment. Despite their clinical success, commercial sealers do not meet the all the requirements of an ideal endodontic sealer, namely the induction capacity of the periradicular regenerative process that requires the recruitment of mesenchymal stem cells (MSC)/osteoblastic precursor cells and their differentiation into osteoblasts for synthesis of the mineralized matrix. Hydroxyapatite nanoparticles (NHAp) present similarities in size, crystallography, and chemical composition to the bone and teeth enamel. <h3>Methods</h3> The biological profile of NHAp and zinc-containing NHAp (NHAp-Zn), tested alone or added to the eluates of the endodontic sealer AH Plus, was characterized taking advantage of the biocompatibility of NHAp, the osteogenic effect of NHAp and zinc, together with the excellent profile of AH Plus. For this purpose, different cells were used: human mesenchymal stem cells derived from Wharton's jelly (hMSC-WJ) for cytotoxicity and osteogenesis assays; CHO-K1 and FMM1 cells for genotoxic and oxidate stress analysis; four oral bacterial and four fungal strains for antimicrobial studies by the minimum inhibitory concentration method. <h3>Results</h3> MTT results showed that AH Plus eluates were toxic, however, there was an excellent recovery of cell viability in the presence of NHAp, with and without zinc, with the most diluted eluate. The mineralization assay showed improved osteogenesis with NHAp, especially in the osteogenic medium, with NHAp-Zn3 in the lowest concentration more effective than NHAp-Zn5. Regarding genotoxicity through micronucleus assay, the results showed no differences between the cell lineages used, with an increase in the frequency of MN only for the most diluted eluate alone and in conjunction with NHAp-Zn3 at the highest concentration. The combination with the best osteogenic potential (most diluted eluate of AH Plus with NHAp-Zn3 at the lowest concentration) and the one recommended in the study did not lead to changes in the frequency of micronuclei, nuclear buds and nucleoplasmic bridges. Antimicrobial assays revealed some of the combinations were bacteriostatic and fungistatic. <h3>Conclusion</h3> All the aforementioned results suggest that NHAp improves osteogenesis in in osteogenic medium without showing genotoxicity indicating that they may be an alternative for bone regeneration therapies in endodontic treatment.