Essential oils (EOs) from Origanum vulgare subsp. vulgare (OVV) and O. vulgare subsp. hirtum (OVH) were evaluated for antioxidant activities (DPPH, ABTS, FRAP, CUPRAC, β-carotene/linoleic acid, phosphomolybdenum and metal chelating), antimicrobial and inhibitory properties against acetylcholinesterase, butrylcholinesterase, tyrosinase, α-amylase and α-glucosidase. Thymol and linalool were identified as major component in OVV and OVH essential oils, respectively. O. vulgare subsp. vulgare exhibited a strong free radical scavenging, reducing power, antimicrobial and acetylcholinesterase, butrylcholinesterase, α-amylase and α-glucosidase inhibitory activity. Interestingly, OVH possess a significant metal chelating and tyrosinase inhibitory activities. The EOs of both species exhibited moderate antibacterial and antifungal activities. The MIC values of the EOs ranged from 85.3 to 426.7. μg/mL. O. vulgare subsp. vulgare showed higher activity against Sarcina lutea with the lowest MIC (85.3. μg/mL), whereas OVH indicated strong activity for Candida albicans with MIC value of 85.3. μg/mL. The data suggest that the Origanum EOs could be used as valuable new natural agents with functional properties for food and pharmacology industries.