In this study, the physicochemical properties, fatty acid composition, antioxidant activities, and in vitro as well as in vivo toxicological safety of emu oil were investigated. Emu oil was shown to have a low acid and peroxide value, low amounts of carotenoid and phenolic compounds, and high doses of oleic acid and linoleic acid. Furthermore, in a bacterial reverse mutation assay, emu oil demonstrated no change in the amount of revertant colonies for all strains. In a chromosomal assay, no aberrations occurred in any of the emu oil treatment groups (1.25, 2.5, and 5 μg/mL). In the bone marrow micronucleus test, emu oil up to 20 mL/kg showed no significant increase in the incidence of micronucleated polychromatic erythrocytes. Moreover, emu oil up to 19.3 mg/kg body weight did not affect body weight in an acute oral toxicity study. These results are crucial for the adoption of emu oil as an alternative source of edible oil.