Extracts of the aerial parts and roots of the wild-growing medicinal plant Gentiana asclepiadea were analysed for their antimicrobial, antibiofilm, and antioxidant activity with quantification of the total phenolic and total flavonoid content. Antimicrobial activity was tested against pathogenic and spoilage bacteria, yeasts, and moulds using the microdilution method. The strongest antibacterial activity was detected on Bacillus species, where minimum inhibitory concentrations (MICs) of from 0.16 mg/mL to 5 mg/mL were obtained, while antifungal activity was low to moderate, with MICs between 1.25 and 20 mg/mL. In the crystal violet assay, the extracts inhibit 50% biofilm formation in the concentration range of from 2.12 to 37.04 mg/mL. Staphylococcus aureus, S. aureus ATCC 25923, and... Pseudomonas aeruginosa ATCC 27853 biofilms were the most sensitive to the presence of extracts. The extracts rich in phenolic compounds showed good DPPH-scavenging activity, with EC50 values between 181.3 and 614.3 μg/mL for extracts of aerial parts and from 426.67 to >1000 μg/mL for root extracts. Even though G. asclepiadea has long been traditionally used, its biological activity is still insufficiently explored, so the obtained results are significant for contributing new knowledge about the plant’s medicinal properties.