Biocompatibility, dimensional stability, radiopacity, flow, and low solubility are the characteristics of an ideal endodontic sealer. This study evaluated and compared in vivo and in vitro biological and physicochemical properties of calcium silicate‐based sealers: Sealer Plus BC (BC), MTA Fillapex (MF); and resin‐based sealers: AH Plus (AHP) and Sealer Plus (SP). Apical papilla cells were exposed to sealer extracts and subjected to MTT, SRB, scratch, alkaline phosphatase enzyme activity (ALP) and Alizarin red staining (ALZ) assays. Sealers were histologically evaluated in connective tissue of Wistar rats in different periods. Radiopacity, film thickness, flow, setting time, pH and element analyses were investigated. BC had better results compared to AHP and MF at hour 72 for MTT assay (p < .05), and the highest cell viability under SRB (p < .05). All sealers presented ALP activity. BC presented the highest mineralized deposition under ALZ (p < .05). BC and MF promoted wound healing. All sealers induced an initial inflammation reaction that decreased over time. Eosinophils were observed at day 7 in MF (p < .05). Despite MF did not present final setting time, the sealers properties were in accordance to ISO 6876/2012 and ASTM C266‐08. All sealers presented cell viability and biocompatibility. BC presented higher pH values and bioactivity. The materials tested showed physico‐chemical properties in accordance with standards, except for MF setting time.