Chlorhexidine (CHX) is considered the gold standard in the antiseptic treatment of the oral cavity, due to its high antibactericidal capability. With the use of CHX mouth-rinse formulations, the bacteriostatic effects are maintained by the adsorption and prolonged release of CHX from oral surfaces. It was believed that antiplaque formation ability and the lack of systemic toxicity of CHX render it an excellent antiseptic in post-surgical dental treatment. However, recent studies have demonstrated that CHX exerts cytotoxic effects on human periodontal tissues, such as gingival fibroblasts and other cells. It also reduces gingival fibroblast adhesion to fibronectin and prevents fibroblast attachment to root surfaces, thus interfering with periodontal regeneration. In this study, using human gingival fibroblasts (HGFs), we investigated effects of CHX on the growth, morphology and proliferation of HGFs. We found that a low concentration (0.002%) of CHX does not interfere with the proliferation and morphology of HGFs. However, a higher concentration (≥0.04%) of CHX inhibits cell proliferation and to a certain extent, affects cell morphology in a time-dependent manner. A decrease in the percentage of cells in the G0/G1 phase and the accumulation of cells in the S phase following treatment with CHX also occurred in a dose-dependent manner. We thus concluded that CHX only at the concentration of 0.002% does not interfere with HGF growth, that is so critical to wound healing. Thus, the application of CHX in the post-surgical antiseptic treatment of the oral cavity should be limited.