Origanum haussknechtii Boiss. (Lamiaceae), an endemic herb growing in Turkey, is consumed as a tea in folk medicine. This study aimed to investigate antimicrobial, antibiofilm, and cytotoxic activities of the methanol, and water extracts from the aerial parts of O. haussknechtii, as well as to determine their phenolic profiles by using HPLC analysis. The methanol extract of O. haussknechtii displayed the highest antimicrobial activity against Escherichia coli with an inhibition zone of 11.57 mm, and MIC of 100 µg/mL. The methanol extract also exhibited an 80.53% inhibition effect on biofilm formation of Pseudomonas aeruginosa. In addition, the methanol extract showed cytotoxicity against the A549 cancer cell line with the growth inhibition of 47.7%. In the phenolic analysis, rosmarinic acid was detected to be a major compound in both methanol, and water extracts, with the amount of 43.17, and 17.68 mg/g extract, respectively. To the best of our knowledge, the present study is the first report on the antimicrobial, antibiofilm, and cytotoxic activities of O. haussknechtii, as well as the phenolic compositions using HPLC. Our data revealed that O. haussknechtii has the potential to be used in pharmaceutical, and cosmetic raw materials, as well as food supplements for application as novel bioactive agents.