Periodontal diseases are dysbiotic conditions in the gingival margin, which are characterized by an imbalance between subgingival communities and the host immune response. Fibroblast has a significant role in the process of wound healing process which is a response to injury and tissue. Therefore, testing plant extracts is required before using them, which one of them is viability testing based on the ability of the material is safe in the field of dentistry. Viability testing is used to monitor the compounds to verify and decide whether it has an effects on the spread of the cell or shows direct cytotoxic effect to the cell that causes cell death. Indonesia's rivers have plentiful of Water hyacinth which contains phenols, alkaloids, flavonoids, and tannins. This plant can be useful for gingival health. The aim of this study is to examine the effect of Water hyacinth leaf extract on the viability of human gingival fibroblast in vitro. Human gingival fibroblast cell culture was harvested placed on a 96-well microplate. Each well was treated with different concentrations of Water hyacinth leaf extract of 1 mg/ml, 0.5 mg/ml, 0.25 mg/ml, 0.125 mg/ml, 0.0625 mg/ml, 0.0312 mg/ml, and 0.0156 mg/ml for 24 hours incubated. MTT assay was performed to evaluate the cell viability. Water hyacinth leaf extract at concentration<0.125 mg/ml did not affect the viability of the cells. It was concluded that Water hyacinth leaf extract was able to change the gingival fibroblast viability, and this effect was concentration dependent. Water hyacinth leaf extract has viability towards human gingival fibroblast at concentration of 0.125 mg/ml, 0.0625 mg/ml, 0.0312 mg/ml and 0.0156 mg/ml. The result suggests that water hyacinth leaf extract has the optimum viability towards human gingival fibroblast cells (75.98%) at concentration of 0.0156 mg/ml.