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Background: Indonesia is one of rich country with large of natural resources where beneficial for the health sector, especially in the pharmaceutical. Many studies have proven the efficacy of Indonesian medicinal plants in the pharmaceutical field, one of them was tobacco which has activity against Candida albicans. Objective: Aims of this study to determine the physicochemical parameters of tobacco leaf extract and its activity against Candida albicans. Materials and Methods : Tobacco (Nicotiana tanabum var. Virginia) leaves were obtained from Ponorogo. Leaves extracted with ethanol 96% solvent by heat reflux method. Extraction results were calculated for yield, water content, ash content, total flavonoid content and total phenol content. Then evaluation its activity against Candida albicans. Result : The results obtained that yield of extract were 23.04% b/b, water content 16.43%, 7.56% ash content, total flavonoid content 2.39 mgQE/g Extract, total phenolic content of 153.74 mgQE/g Extract. From the GCMS analysis, it was found that the nicotine content was 49.18%. The activity against Candida albicans was tested with macrodilution methode in parallel concentrations of 100.000 µg/mL -195.31 µg/mL and the result showed that there is no activity against the tested antifungal. Conclusion : the study showed that pyridine was one of major compound where detected with GC-MS and there is no activity of Nicotiana tabacum var Virginia against Candida albicans.