The aim of this study was to investigate the cytotoxicity of modified nonequilibrium plasma with chlorhexidine digluconate (CHX) on human gingival fibroblasts (HGFs), and to evaluate the biosecurity of modified nonequilibrium plasma with 2% CHX as a new method of root canal treatment. Tissue samples taken from human gingiva were primarily cultured and passaged. Cells from passages 3-7 were used. HGFs were treated by modified nonequilibrium plasma with 2% CHX for 0 min (control group), 30 s, 1 min, 1.5 min, 3 min, 5 min, and 10 min, respectively, and then they were incubated for 0, 24, and 48 h. After that, cell counting kit-8 (CCK-8) assay was applied to analyze the cytotoxicity of modified nonequilibrium plasma with 2% CHX on HGFs. There was no significant difference between the 0 h group treated with the modified nonequilibrium plasma for 1 min and the control group (P > 0.05). However, there were significant differences between all the other treated groups and the control group (P < 0.05). When treated for 1.5 min or shorter, the cell viability was obviously increased; while treated for 3 min or longer, it was obviously reduced. Moreover, when successively cultured for 0, 24, and 48 h, cell viability was decreased at first and then increased in the 3-min-treated and 5-min-treated groups. The modified nonequilibrium plasma with 2% CHX was of no influence on cell viability in 1.5 min treatment, and it could be safely used on root canal treatment.