The phenomenon of using plants for therapeutic purposes dates back to the earlier times in the history of humanity. Even though there have been remarkable developments in the modern medicine, pharmaceutical, and chemical industries, the therapeutic use of medicinal plants is still up to date. Due to the emergence of antibiotic-resistant strains and the proliferation of side effects in synthetic drugs, the importance of research on the antimicrobial potential and biochemical composition of plant extracts has increased. The main purpose of this present study is to screen both the antibacterial and antifungal activity of endemic Helichrysum arenarium ssp. aucheri ethanol extract by disk diffusion on some Gram-negative and Gram-positive bacteria and Candida albicans, and to identify the major compounds found in the ethanol extract by GC/MS. According to the results obtained from the study, it can be proposed that the extract showed antibacterial and antifungal activity for all microorganisms excluding Salmonella typhimurium SL1344, Salmonella infantis, E. coli (food isolate), Escherichia coli ATCC 25922 and Enterobacter aerogenes ATCC 13048, and some chemical compounds were detected, which may be responsible for the antimicrobial activity.