B Background: kombucha is a fermented beverage with a low ph that has the potential to cause tooth enamel demineralization through the dissolution of hydroxyapatite minerals. this process may increase calcium release and reduce enamel hardness. objective: this study aimed to determine the effect of kombucha beverage on calcium release and enamel hardness. methods: this study was an in vitro laboratory experimental study using a post-test-only control group design. the samples were divided into a treatment group exposed to kombucha and a control group exposed to distilled water. calcium release was measured using a uv–vis spectrophotometer, while microhardness values were measured using the vickers hardness test. the data were analyzed using statistical tests based on the data distribution, with a significance level of p < 0.05. results: the mean calcium release in the kombucha group (0.1326) was higher compared with the distilled water group (0.0071), indicating that acid exposure from kombucha induced enamel demineralization. statistical analysis showed a significant difference in calcium release between the two groups (p < 0.05). the mean microhardness value was 198.2 vhn in the kombucha group and 219.6 vhn in the control group. descriptively, enamel hardness was lower in the kombucha group however, the difference was not statistically significant (p > 0.05). conclusion: in this study, kombucha beverage increased calcium release as an indicator of enamel demineralization but did not have a significant effect on enamel microhardness.