OBJECTIVE The aim of the present study is to evaluate the biocompatibility of glass ionomer cements (GICs) with and without chlorhexidine (CHX) as well as coated with varnish or not using in vitro cytotoxicity test. METHODS Biocompatibility of Fuji IX, Fuji IX with varnish, Fuji IX with 1% CHX diacetate and Fuji IX with 1% CHX diacetate with varnish was determined with in vitro cytotoxicity assay by using L929 mouse connective tissue fibroblasts. After 72 h, cell viabilities were evaluated by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay to determine the effects of the cements on the mitochondrial function and microscopic images were taken by scanning electron microscopy. RESULTS Statistical analysis was performed by one-way analysis of variance followed by the Bonferroni post-hoc test at a significance level of P < 0.05. 72 h after treatment, there were statistically significant differences between Fuji IX and Fuji IX-CHX (P < 0.001). In addition, the reduction of the cytotoxicity by coating the GICs with varnish was indicative and increased the cell viability ratio (P < 0.001). CONCLUSIONS Fuji IX coated with varnish was found to be the most biocompatible one among others. Thus adding CHX significantly reduced the cell viability, it is assumed that, due to the leakage of CHX and the other components of the GICs to the cell culture medium, the cell viabilities were decreased, so it is highly recommended to use varnish not only to reduce the water loss from the GICs, but also to reduce the cytotoxicity of the GICs.