The essential oils (EOs) were isolated by hydrodistillation from wild and cultivated Pistacia lentiscus L. var. chia—mastic gum tree (Anacardiaceae) from two natural habitats, namely from Cesme–Uzunkoy (1) and Mordogan (2), and one cultivated source, Cesme–Germiyan (3), in Izmir, Turkey. This comparative study evaluated the chemical composition and biological activity of mastic gum essential oils (MGEOs). For this purpose, MGEOs 1–3 were analyzed by gas chromatography–flame ionization detection (GC-FID), gas chromatography–mass spectrometry (GC-MS), and chiral GC for α-pinene. Laboratory assays were conducted to assess for potential in vitro cytotoxicity (multiple in vitro cancer cell lines), antimicrobial properties (five bacterial species and yeast), anti-inflammatory activity (inhibition of inducible nitric oxide synthase, iNOS), and the attraction of Ceratitis capitata (Mediterranean fruit fly, medfly), respectively. Chemical analysis indicated that MGEOs 1 and 2 were rich in α-pinene (56.2% and 51.9%), myrcene (20.1% and 18.6%), and β-pinene (2.7% and 3.1%), respectively; whereas MGEO-3 was characterized by a high level of α-pinene (70.8%), followed by β-pinene (5.7%) and myrcene (2.5%). Chiral GC analyses showed that concentration ratios between (−)/(+)-α-pinene and (−)-α-pinene/myrcene allowed for differentiation between wild and cultivated MGEO sources. In biological assays, MGEOs 1–3 did not exhibit significant antimicrobial effects against the pathogens evaluated and were not strong attractants of male medflies; however, all three MGEOs displayed a dose-dependent inhibition of iNOS, and MGEOs 1 and 2 exhibited selective in vitro cytotoxicity against human cancer cells. These results suggest that wild-type mastic gum oils from Cesme and Mordogan (MGEOs 1 and 2) are potential sources of beneficial products and warrant further investigation.