Olive oil application in the cosmetic industry may be extended by its ozonation, bringing about new oil properties and increased stability. Olive oil treated with 0.04 mole O3 or 0.10 mole O3 per 100 g oil was subjected to chemical parameters evaluation and composition scrutinizing by gas chromatography–mass spectrometry (GC-MS) and headspace solid-phase microextraction (HS-SPME) GC-MS analysis. The biological activity of refined and ozonated oil included their antimicrobial properties by the agar diffusion method and cytotoxicity by the MTT assay towards two normal (LLC-PK1, HaCaT) and two cancerous (Caco-2, HeLa) cell lines. The oils served as the basis in cosmetic emulsions. The chosen organoleptic features, preservative efficacy in a challenge test, and persistency during six months of these formulations were assessed. However, the ozonation of the olive oil resulted in a decrease in unsaturated acids; several additional compounds were detected in the ozonated oil, which positively affect the physicochemical, sensory, and functional properties of cosmetic emulsions. Emulsions based on the ozonated olive oil retain their properties longer compared to emulsions based on the refined olive oil. Ozonated oil treated with 0.10 mole O3/100 g oil allowed increasing the shelf life of the non-preserved formulation up to six months. A weak inhibitory effect against Candida albicans and Aspergillus brasiliensis was also demonstrated for this emulsion in the challenge test. Moreover, an interesting aroma, slightly enhanced antimicrobial activity against Escherichia coli, Staphylococcus aureus, C. albicans, A. brasiliensis, and a lack of cytotoxicity at concentrations 625 µg mL−1 make the ozonated olive oil a promising raw material for the cosmetics and pharmaceutical industries.
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