The Phlomis kurdica Rech.fil. (Lamiaceae) essential oil was investigated for its chemical constituents and in vitro biological activities. Volatile components were characterized, antimicrobial and anticholinesterase activities were performed. The oil was analyzed both by gas chromatography-mass spectrometry and gas chromatography-flame ionization detector, simultaneously. The in vitro antimicrobial microdilution method was used against Bacillus cereus ATCC 14579, Pseudomonas aeruginosa ATCC 10145, Escherichia coli NRLL B-3008, Staphylococcus aureus ATCC 6538, Helicobacter pylori ATCC 43504, Staphylococcus epidermidis ATCC 14990, and Candida albicans ATCC 90028, respectively. Additionally, the cholinesterase enzyme inhibition activities were tested in vitro using the Ellman method. A total of forty-four different volatile constituents were identified, where the main components were characterized as germacrene D (55.4%), (Z)-beta-farnesene (11.2%), and hexadecanoic acid (8.4%), respectively. The P. kurdica oil showed moderate inhibition of cholinesterases, where inhibitory antimicrobial activity was against B. cereus among the tested microorganisms.