This study investigates the development of an oil-in-water (O/W) emulsion enriched with a high concentration of ostrich oil, recognized for its abundant content of oleic acid (34.60 ± 0.01%), tailored for skincare applications. Using Span and Tween emulsifiers, we formulated an optimized emulsion with 20% w/w ostrich oil and a 15% w/w blend of Span 20 and Tween 80. This formulation, achieved via homogenization at 3800 rpm for 5 min, yielded the smallest droplet size (5.01 ± 0.43 μm) alongside an appropriate zeta potential (−32.22 mV). Our investigation into the influence of Span and Tween concentrations, types, and ratios on the stability of 20% w/w ostrich oil emulsions, maintaining a hydrophile–lipophile balance (HLB) of 5.5, consistently demonstrated the superior stability of the optimized emulsion across various formulations. Cytotoxicity assessments on human dermal fibroblasts affirmed the safety of the emulsion. Notably, the emulsion exhibited a 52.20 ± 2.01% inhibition of linoleic acid oxidation, surpassing the 44.70 ± 1.94% inhibition observed for ostrich oil alone. Moreover, it demonstrated a superior inhibitory zone against Staphylococcus aureus (12.32 ± 0.19 mm), compared to the 6.12 ± 0.15 mm observed for ostrich oil alone, highlighting its enhanced antioxidant and antibacterial properties and strengthening its potential for skincare applications. The optimized emulsion also demonstrates the release of 78.16 ± 1.22% of oleic acid across the cellulose acetate membrane after 180 min of study time. This successful release of oleic acid further enhances the overall efficacy and versatility of the optimized emulsion. Stability assessments, conducted over 6 months at different temperatures (4 °C, 25 °C, 45 °C), confirmed the emulsion’s sustained physicochemical and microbial stability, supporting its promise for topical applications. Despite minor fluctuations in acid values (AV) and peroxide values (PV), the results remained within the acceptable limits. This research elucidates the crucial role of emulsification in optimizing the efficacy and stability of ostrich oil in skincare formulations, providing valuable insights for practical applications where stability is paramount.