Chios mastic gum (CMG), the resin of Pistacia lentiscus var. Chia, is a product with great ethnopharmacological and economic significance. This study attempts to investigate, for the first time, the activity of CMG, its fractions and isolated compounds against specific enzymes, which play pivotal roles in the degradation of proteins contained in skin connective tissue. Initially, crude CMG was subjected to extraction, fractionation and isolation through different chromatographic techniques to obtain the acidic and neutral fraction of terpenes. Additionally, the characteristic and major active triterpene acids of CMG, masticadienonic and isomasticadienonic acids (MNA, IMNA) were isolated in pure form. All samples were analysed by means of High-Performance Thin-Layer Chromatography (HPTLC) with four distinct development systems to obtain their constituents’ profile. Finally, samples were tested for their ability to inhibit the elastase and collagenase enzymes. According to our findings, for collagenase, a mixture of MNA and IMNA demonstrated the most potent activity with an IC50 value of 31.07 μg/mL, while for elastase CMG’s acidic fraction provided the most promising results with an IC50 value of 17.30 μg/mL. Overall, these results attempt to fill the gap in scientific knowledge about the use of CMG and its constituents in skincare and cosmetic products.