Fungal infections are a serious global health concern, contributing significantly to morbidity and mortality rates. The emergence of antifungal resistance and the relative toxicity of existing medications have made it necessary to explore alternative treatments, such as herbal medicines used in traditional folk therapies. This study aims to evaluate the in vitro antifungal activity of Dactylorhiza hatagirea, a Himalayan orchid. The roots of the plant were collected and extracted using a hydroalcoholic solvent. Qualitative and quantitative analyses were performed, and antifungal activity was assessed. The results showed the presence of flavonoids, phenols, proteins, carbohydrates, saponins, and tannins. The total phenol and flavonoid content were found to be 0.496mg and 0.784mg/100mg, respectively. The extract with a concentration of 100mg/ml demonstrated a zone of inhibition of 13±0.86mm against Candida albicans and 10±0.94mm against Aspergillus flavus. Therefore, the study concludes that the roots of Dactylorhiza hatagirea possess potent antifungal activity