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This study aimed to evaluate the spasmolytic activity of an underground parts extract of Gentiana asclepiadea L. (Gentianaceae), assess its antioxidant and antimicrobial activities, and explore the impact of extract encapsulation on the aforementioned bioactivities. An extract encapsulated by spray drying with whey protein, pure extract, and pure whey protein were comparatively tested. The main compounds identified via HPLC-DAD analysis underwent in silico ADME assessment. The spasmolytic effect was tested on a model of spontaneous rat ileum contractions, and the mechanism of action was further evaluated on acetylcholine-, KCl-, CaCl2-, BaCl2-, histamine-, N(ω)-nitro-L-arginine methyl ester-, and glibenclamide-modified contractions. The most abundant compounds were secoiridoids (dominantly gentiopicroside), followed by C-glycosylated flavonoids and xanthones. Both pure and encapsulated extracts achieved significant spasmolytic effects, despite the spasmogenic activity of pure whey protein. The extract may exert its spasmolytic effect through multiple pathways, predominantly by antagonizing the Ca2+ channel and opening the K+ channel, while the nitric oxide pathway appears not to be involved. The antimicrobial and antioxidant activities of the pure extract were moderate. The extract stabilized by encapsulation retained all of the tested bioactivities of the unencapsulated extract. The obtained results suggest that G. asclepiadea has potential for use in the treatment of some gastrointestinal complaints and that the encapsulated extract could be a valuable functional ingredient in pharmaceutical and food products.