Salvia aethiopis L., a member of the Lamiaceae family, has gained attention due to its bioactive compounds with potential therapeutic applications. This study investigated the chemical composition and biological activities of S. aethiopis ethanol extract collected from Karaman province, Türkiye. The phenolic profile of the plant was characterized using LC–MS/MS analysis, which revealed substantial amounts of rosmarinic acid, ferulic acid, caffeic acid, and p-coumaric acid. The antimicrobial activity of the extracts was evaluated against various Gram-positive and Gram-negative bacteria, as well as fungal strains, using the agar well diffusion method. The results demonstrated potent antibacterial effects, particularly against Staphylococcus aureus, Micrococcus luteus, and Klebsiella pneumoniae. Furthermore, the cytotoxic potential of S. aethiopis was assessed on H-460 non-small cell lung cancer cells. The extract exhibited a dose-dependent cytotoxic effect, with a significant reduction in cell viability after 24 and 48 h of treatment, yielding IC50 values of 80.08 and 56.19 μg/mL, respectively. These findings suggest that S. aethiopis possesses promising antimicrobial and anticancer properties, which could contribute to the development of novel therapeutic agents.