The genus Gentiana (Gentianaceae) with approximately 400 species is distributed in the temperate regions of Asia, Europe, and America. It is represented by 12 species in Turkey [1]. Gentiana species are used in herbal medicine to treat digestive problems, fever, hypertension, muscle spasms, wounds, cancer, sinusitis, and malaria [2]. In this present study, the methanolic extract of the aerial parts of Gentiana pyrenaica L. was investigated for its biological activities such as antioxidant, acetylcholinesterase (ACE), butyrylcholinesterase (BCE), monoamine oxidase MAO-A, MAO-B, cyclooxygenase COX-1, COX-2 inhibitory effects and for its total phenolic content using Folin-Ciocalteu assay. According to the antioxidant activity results, G. pyrenaica showed weak ferric-reducing power with a EC50 value of 1.84 and ABTS radical cation decolorization activity with an inhibition of 20.27% when compared to the standards BHT (EC50 = 0.2) and Trolox (79.01%) respectively at 0.5 mg/mL concentration. Chelating ability of G. pyrenaica was 39.51% whereas EDTA showed 78.87% chelating activity at 0.2 mg/mL. Also G. pyrenaica showed weak 26.04% ACE, 14.02% BCE, strong 75.18% MAO-A and 75.90% MAO-B, 18.16% COX-1, 27.42% COX-2 inhibitory activities at 0.25 mg/mL. The total phenolic content of G. pyrenaica was 208.46 mg gallic acid equivalent/100 g plant.