Camphorated phenol, chlorocamphene, formocresol and eugenol are volatile antimicrobial agents, generally used for root canal treatment. The volatility has a toxic effect to surrounding tissue, such as alveolar bone cells. Objective: To evaluate the toxicity of four intracanal medication on cultured mouse osteoblast cell line (MC3T3-E1). Method: MCT3T3-E1 cells were fed with á-MEM as control group. Cultured were treated with clinical concentration and 2, 4, 6, 24 and 96 fold dilution of camphorated phenol, chlorocamphene, formocresol, eugenol. Following 24 hours of treatment, cultures were analyzed by MTT assay, TUNEL assay and stained with trypan blue to test cytotoxicity, apoptosis and morphological changes, respectively. Results: This study showed that cells exposed to these substances with all tested concentrations have cytotoxic index more than IC95 except for at 96 fold dilution concentration. At 96 fold dilution concentration, cells show 65.22%, 60.87%, 41.30%, 45.65% viability when tasted with camphorated phenol, chlorocamphene, formocresol and eugenol, respectively. Morphological and TUNEL assays indicated that cells treated with 96 fold dilution concentration were undergone apoptosis. Conclusion: The intracanal medication exerts potential harmful to bone cell. Hence, the cytotoxic effect of these should be concerned and carefully used during clinical endodontic procedures.