Fraxinus angustifolia subsp. angustifolia is a plant with an age-old use for the production of manna. However, it is also a valuable source of fixed oil rich-seeds. In the present study we examined the chemical and biological properties of this oil in order to support a possible application in foodstuffs, nutraceuticals and cosmetics. Fatty acid composition, volatile and phenolic substances were evaluated. Oleic and linoleic acid represented 45.5% and 50.0%, respectively, of the total fatty acid composition. Among polar phenolic substances identified (secoiridoids, phenylethanoid glycosides, phenolic acids and alcohols, flavonoids, coumarins) isoverbascoside is for the first time reported in this species. Volatiles were mainly characterized by sesquiterpenes. The oil showed good antioxidant activity, in terms of ABTS radical scavenging activity, with an IC50 value of 28.2 μg/mL. The antiproliferative activity was also investigated: amelanotic melanoma (C32) and lung carcinoma (A549) cells were the most sensitive with IC50 values comparable to that of the positive control vinblastine. These findings shed light on the potential use of F. angustifolia subsp. angustifolia fixed oil in nutraceutics and cosmetics.