The aim of this study is to investigate the antimicrobial compounds present in the lichen Usnea albopunctata. Ethyl acetate extract was purified by silica gel column chromatography to obtain a major compound and the chemical structure was characterized by 1H-NMR, 13C-NMR, DEPT, 1H-1H COSY, HMQC, HMBC, UV, and HR-MS spectroscopic methods as protolichesterinic acid. The antimicrobial activity was estimated by determination of the minimal inhibitory concentration by the broth microdilution method and agar disc diffusion method against thirteen human pathogenic bacterial and four fungal strains. Protolichesterinic acid recorded significant broad spectrum antimicrobial property. The best antibacterial activity was recorded against K. pneumonia (0.25？μg/mL) and V. cholerae (0.5？μg/mL). Significant antifungal activity was recorded against T. rubrum (0.12？μg/mL), which is significantly better than the standard antifungal agent. Protolichesterinic acid is reported for the first time from Usnea albopunctata. Antifungal activity of protolichesterinic acid against medically important fungi is also reported for the first time. Thus the results of the present study suggest that protolichesterinic acid has significant antimicrobial activities and has the strong potential to be developed as an antimicrobial drug after further clinical evaluation. 1. Introduction In recent years, both in practice and in theory, there has been great interest in new preparations of natural origin for the control and prevention of various human, animal, and plant diseases. It is known that the long-term use of synthetic drugs can cause numerous side effects and sometimes resistance [1]. Unlike synthetic drugs, bioactive natural products have beneficial effects on the whole organism. Infectious diseases are one of the world’s leading causes of premature deaths [2]. Antibiotics which are widely used for the treatment of infectious diseases are under constant threat due to the emergence of antibiotic resistant pathogens such as methicillin resistant Staphylococcus aureus (MRSA), multidrug resistant Pseudomonas aeruginosa, vancomycin resistant Enterococcus (VRE), and multidrug resistant Mycobacterium tuberculosis [2–7]. Hence, search for novel antimicrobial compounds or alternative therapy for these infections is inevitable. In the search for new bioactive preparations of natural origin, lichens are a subject of interest to many research teams. Lichens produce secondary metabolites known as “lichen substances” and more than one thousand secondary metabolites, mainly monoaromatics, depsides,
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