The aim of this study was to evaluate the influence of [2-(methacryloyloxy)ethyl] trimethylammonium chloride (METAC) in the physico-chemical properties, antibacterial activity and cytotoxicity of an experimental resin-based sealant.An experimental resin-based sealant was formulated with dimethacrylates and a photoinitiator system. METAC was added at 2.5 wt.% (G) and 5 wt.% (G) into the experimental resin-based sealant, and one group remained without METAC as control (G). The resin-based sealants were analysed for polymerization behaviour and degree of conversion (DC), Knoop hardness (KHN) and softening in solvent (ΔKHN), ultimate tensile strength (UTS), contact angle, surface free energy (SFE), immediate and long-term micro-shear bond strength (μ-SBS) and antibacterial activity and cytotoxicity against human keratinocytes.The experimental resin-based sealants presented different polymerization behaviours without significant differences in the DC (p > 0.05). There was no significant difference for initial KHN (p > 0.05). The ΔKHN ranged from 51.62 (±3.70)% to 62.40 (±4.14)%, with higher values for G (p < 0.05). G and G had decreased μ-SBS between immediate and long-term tests (p < 0.05) without significant differences among groups in the immediate and long-term analyses (p > 0.05). There were no significant differences for UTS, contact angle and SFE among groups (p > 0.05). G and G presented immediate and long-term antibacterial activity (p < 0.05) without cytotoxicity compared to G (p > 0.05).The addition of METAC provided antibacterial activity to the experimental resin-based sealant.METAC is an effective quaternary ammonium compound as an antibacterial agent for resin-based sealants without cytotoxic effects against human keratinocytes.