The antimicrobial activity and possible synergistic effects of extracts and compounds isolated from Usnea steineri were evaluated against four resistant bacterial species. A phytochemical study of the acetone extract of U. steineri resulted in the isolation and characterization of difractaic acid and (+)–usnic acid as the main compounds. The acetone extract showed strong activity (less than 10 μg/mL) against resistant strains of Staphylococcus epidermidis and Enterococcus faecalis, and (+)–usnic acid exhibited strong activity against S. epidermidis (MIC 3.12 μg/mL), S. aureus and S. haemolyticus (MIC 12.5 μg/mL). Combinations of penicillin and tetracycline with (+)–usnic acid did not show any synergistic antimicrobial effects. Difractaic acid was inactive. Our results showed that the acetone extract of U. steineri possesses significant in vitro antimicrobial activity, which is likely related to the presence of (+)–usnic acid.