Continuing our work on the sources of natural bioactive compounds, we evaluated the antimicrobial and antioxidant activities of Nepeta trachonitica as well as its major phenolic content using the high-performance liquid chromatography-mass spectrometry/mass spectrometry (HPLC-MS/MS) technique. For antioxidant activity, ferric reducing antioxidant power (FRAP) and cupric ion reducing antioxidant capacity (CUPRAC) methods were performed to measure the reducing power and 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay was employed to evaluate the radical scavenging activity of the sample. For antimicrobial activity, three Gram-positive and four Gram-negative microbial species as well as three fungi species were tested. N. trachonitica appeared to have reasonable antioxidant activity and decent antimicrobial activity as indicated by the inhibition of the organisms' growth. The most susceptible species were Bacillus subtilis ATCC 6633 and Escherichia coli ATCC 11229 among the organisms tested. Ethanol extract of the plant has the highest effect on Saccharomyces cerevisiae but no effect on Yarrowia lipolytica. The HPLC-MS/MS analysis showed that at least 11 major phenolic compounds of N. trachonitica exist, the major ones being rosmarinic acid, chlorogenic acid and quinic acid. The obtained results suggest that N. trachonitica could be a promising source for food and nutraceutical industries because of its antimicrobial and antioxidant properties and phenolic compounds.