INTRODUCTION: The aim of this study was to compare the cytotoxicity of ProRoot MTA (PMTA), Root MTA (RMTA) and Portland cement (PC) on human gingival fibroblasts (HGFs). MATERIALS AND METHODS: The extracts of the test materials were placed close to human gingival fibroblasts (HGFs) within 96-well plates. Cellular viability was assessed using MTT assay in different intervals (freshly mixed, 4, 24, and 168 hours after mixing). The data were analyzed using the One-way ANOVA and Tukey’s test at 95% significance level. RESULTS: It was indicated that there was not a significant difference in cytotoxicity of test materials (P>0.05). In addition, there was not a statistically significant difference between different time intervals within each group (P>0.05). CONCLUSION: PMTA, RMTA and PC showed comparative biocompatibility while evaluated in-vitro.