The purpose of the present study was to compare the biological and the physico-chemical properties of bioceramic-based root canal sealers, calcium hydroxide-based, MTA-based and epoxy resin-based root canal sealers. Two bioceramic-based sealers, one calcium hydroxide-based sealer, one MTA-based sealer and two epoxy resin-based sealers were tested. EasySeal and MTA Fillapex showed severe citotoxic activity, AH Plus and SealapexTM moderate cytotoxicity, BioRoot? RCS and TotalFill BC Sealer were both cytocompatible. Except for TotalFill BC Sealer, all root canal sealers caused inhibition zones when tested with E. faecalis. The highest inhibition zone was observed for EasySeal, followed by AH Plus. BioRoot? RCS, SealapexTM and MTA Fillapex showed the lowest inhibition zone. All the tested materials showed different degree of antibacterial activity by using direct contact test (DCT). The highest values were observed for BioRoot? RCS, TotalFill BC Sealer and EasySeal, followed by MTA Fillapex and SealapexTM. Except for BioRoot RCS and TotalFill BC Sealer, all the root canal sealers fulfilled the requirements of the ISO 6876 standard, demonstrating a weight loss less than 3%. Bioroot RCS, TotalFill BC Sealer and SealapexTM exhibited high alkaline pH with an increase both for BioRoot? RCS and TotalFill BC Sealer after 24 hours. The new bioceramic-based sealers showed acceptable physico-chemical properties, but BioRoot? RCS and TotalFill BC Sealer seems to be too soluble, not respecting ISO 6876 requirements.