The essential oil profile of Thymbra spicata L. subsp. spicata L. (Lamiaceae) was analyzed in this study using gas chromatography/mass spectrometry (GC-MS). Additionally, antioxidant and cytotoxic activities, along with inhibition activities for acetylcholinesterase, butyrylcholinesterase, urease, tyrosinase, elastase, collagenase, and angiotensin-converting enzymes, were assessed. Additionally, the acaricidal effects of the essential oil with strong antioxidant properties from T. spicata subsp. spicata, as well as the AgNO3 nanoparticles synthesized using this oil, were evaluated on Hyalomma marginatum nymphs. The main compounds present in the essential oil was identified as carvacrol (71.19%). The species exhibited remarkably high antioxidant activity, particularly in the ABTS cation radical scavenging activity and CUPRAC copper reducing capacity assays, with IC50 and A0.5 values of 1.80±0.053 and 1.30±0.12 μg/mL, respectively. Additionally, the sample exhibited significantly high acetylcholinesterase and butyrylcholinesterase enzyme inhibition activities, with inhibition rates of 76.09±1.63% and 97.94±0.71%, respectively. It was also determined that the essential oil had moderate antimicrobial activity against the bacteria, and MIC values varied between 312 μg/mL and 1250 μg/mL. At the end of 24 hours, The LC50 values of essenttial oil and silver nanoparticles were determined to be 18.48 and 36.30 μg/mL, respectively. The results of this study indicate that T. spicata may find application in dietary supplements due to its high carvacrol content and strong antioxidant, anticholinesterase, and acaricidal activities