One of the largest genus of Lamiaceae, Stachys, extended from south western part of Asia to America, represented in Turkey with % 48 endemism rate. In this presented study LC-ESI-MS/MS properties, anticandidal, antioxidant and cytotoxic activity of methanol extract of an endemic Stachys rupestris Montbret et Aucher ex Bentham was determined. Total phenol content, Trolox Equivalent Antioxidant Capacity assay (TEAC), 1,1-Dipheny1-2-picrylhy-drazyl (DPPH) radical scavenging activity tests were made for antioxidant activity. Phenolic compounds determination were performed with LC-MS/MS. Extract was further investigated against thirteen selected human pathogenic Candida strains. Additionally, cytotoxic effect of the extract was determined against NIH3T3 mouse embryonic fibroblast cell lines by MTT assay. Total phenol content was found as 18.90 +/- 0.54 mg GAE. IC50 value of DPPH radical scavenging activity was determined as 0.04 +/- 0.003 mg/ml. TEAC activity was found as 0.7 +/- 0.02 mM while IC50 value of Gallic acid = 0.002 +/- 0.0001 mg/ml and TEAC of gallic acid 2.47 +/- 0.07 mM. Hydroxybenzoic acid derivatives and flavonoids were identified as main compounds Anticandidal activity of the extract was evaluated with CLSI M27 - A2 protocol against 13 Candida strains. Remarkably, two strains of Candia krusei were inhibited by S. rupestris extract, with the concentrations of 31.25 and 62.5 mu g/mL. Cytotoxicity test showed that S. rupestris extract does not possess a cytotoxic activity against NIH3T3 cell lines at these concentrations.