The potential health benefits of the essential oils of six different populations of Origanum heracleoticum L. from Calabria (Italy) were assessed, together with the evaluation of the influence of the site of collection on their metabolic profile and biological activity. Different terpenoids were identified, being thymol, carvacrol and o-cymene among the most abundant ones. Hierarchical cluster analysis allowed the identification of two main groups, which were discriminated by the altitude of collection. The antioxidant activity was tested by means of two in vitro assays: DPPH and β-carotene bleaching test. Samples EO-4 and EO-3 were the most active in protecting linoleic acid from peroxidation, with IC50 values of 4.00 and 4.68 μg/mL after 30 minutes of incubation. The anti-inflammatory potential was assessed through the evaluation of the capacity to inhibit NO production in lipopolysaccharide (LPS)-induced murine macrophage RAW 264.7 cell line. All essential oils induced a dose-depending inhibitory effect. An excellent activity was demonstrated for sample EO-4, followed by sample EO-3 (IC50 values equal to 32.77 and 49.48 μg/mL, respectively). Observed biological properties were correlated to the phytochemical content of analyzed samples.