Featured Application Ensuring safe food and care for the health of consumers and animal welfare are among the objectives of the EU's policy and food production sectors. Moreover, there is a great need for the introduction of new, environmentally friendly technologies, including the extraction of antimicrobial substances. Supercritical fluid extraction is becoming an increasingly popular method for the recovery of bioactive compounds, representing a non-toxic, cheap, and generally recognized as safe (GRAS) technique, compared to conventional extraction methods, which often require higher temperatures and large amounts of organic solvents. The research presented here is the first to describe the biological activity of Glechoma hederacea extracts obtained by means of supercritical fluid extraction. Therefore, it provides new information and broadens the existing knowledge in the study of the properties of SC-CO2 plant extracts and their potential application. Glechoma hederacea var. longituba is a herbaceous plant from the Lamiaceae family, used in herbal medicine. In this work, we aimed to assess the total phenolic content, antioxidant, antimicrobial and antibiofilm activity of extracts obtained from G. hederacea via supercritical dioxide extraction with methanol as a co-solvent under different extraction conditions. The results showed that the activity of the obtained SC-CO2 extracts is strongly dependent on the extraction temperature. Significantly higher total polyphenol content, as well as antioxidant and antimicrobial activity towards bacteria and yeasts, was observed in the extract obtained at 40 degrees C, compared to extracts obtained at 50 degrees C and 60 degrees C; however, antifungal activity against filamentous fungi was not dependent on the extraction conditions. Antimicrobial activity also depended on the microorganism type. Higher sensitivity was exhibited by Gram-positive bacteria than by Gram-negative bacteria, with S. aureus and P. aeruginosa being the most sensitive species among each group. The most susceptible fungi were Candida albicans and Sclerotinia sclerotiorum. The antibiofilm activity was differentiated and depended on the extraction conditions, the microorganism and the method of biofilm treatment. All tested extracts inhibited biofilm formation, with the extract obtained at 40 degrees C showing the highest value, whereas only extract obtained at 60 degrees C efficiently removed mature biofilm.