The number of cases of fatal infections in humans and animals caused by multiresistant and panresistant bacterial strains has been dramatically increased over the past 10 years on almost every continent. Therefore, implementation of newer technologies in order to develop processes for isolation of substances with strong antibacterial activities from natural sources is of increasing interest. In this study, advantage of high pressure processing and supercritical fluid extraction with carbon dioxide in isolation of active principles from lichen Usnea barbata L. was presented. Supercritical extraction showed extremely higher selectivity toward usnic acid compared to the conventional techniques tested. In order to simulate the process and estimate mass transfer coefficients, the new mathematical model of Sovová (2011) was applied. Supercritical extract obtained at 40 °C and 30 MPa showed stronger antibacterial activity against the most of tested strains compared to pure usnic acid and extracts obtained using ethanol as a solvent. Further, supercritical extract with lower usnic acid content expressed stronger antibacterial activity against the majority of tested strains including MRSA strains compared to the pure usnic acid and the supercritical extract with the higher usnic acid content. The same extract was far more effective than ampicillin against Streptococcus uberis, all tested Enterococcus faecalis, MRSA and MRSA ATCC 33591. Against few tested strains, the supercritical extract was superior to erythromycin and penicillin as well. These findings justified possible benefits of inclusion of supercritical extracts into pharmaceutical and cosmetic formulations. Applied mathematical model described the SFE process with high accuracy. Keywords: Usnea barbata Supercritical fluid extraction Antibacterial activity Usnic acid MRSA.