Since the dawn of time, people have relied on herbal medicine to heal many ailments. Lavandula coronopifolia (LC) is an aromatic plant that has been utilized in ethnomedicine since ancient times. This investigation aimed to figure out the chemical constituents of the volatile oil (VO) of the LC herb from Palestine for the first time and determine its microbicidal, anti-inflammatory, and cytotoxic effects. The LC aerial parts VO phytochemical constituent characterizations were performed utilizing gas chromatography linked to a mass spectrometric system. While, the effects of LC-VO's ability to suppress microbial growth were established against selected bacterial and fungal strains by a broth microdilution technique. Besides, an in vitro cyclooxygenase enzyme (COX) suppressant bioassay kit was utilized to estimate the anti-inflammatory effect against bovine cyclooxygenase (COX) type 1 and 2. In addition, the colorimetric MTS test was employed to determine the cytotoxic activity against the cervical adenocarcinoma (HeLa) tumor cell line. Linalyl acetate (41.65 ± 0.91%) and linalool (41.41 ± 0.77%) were identified as major components of LC-VO, and oxygenated monoterpenoid was the dominant phytochemical class of this VO. The LC-VO demonstrated potent bactericidal and fungicidal effects, especially on methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus aureus, Proteus vulgaris, and Candida albicans (MIC = 0.33 ± 0.03, 0.63 ± 0.1, 1.25 ± 0.81, and 0.16 ± 0.06 µg/mL), respectively. Besides, LC-VO suppressed COX enzymes type-1 and −2, with an inhibition percentage at 50 µg/mL of 95.00 and 88.02 percent, respectively, in comparison with the positive control, ketoprofen non-steroidal anti-inflammatory drugs (NSAID), at the same concentration with an inhibition percentage of 96.25 and 65.02%, respectively. In addition, the LC-VO inhibited the growth of HeLa cells dose-dependently, with an IC50 dose of 0.71 ± 0.01 µg/mL. The current study demonstrates that LC-VO is a promising alternative for the healing or preventing various infectious diseases, cancer, and chronic inflammation. These results reveal that LC-VO has beneficial health benefits and has the potential to be utilized for therapeutic purposes.