Moringa oleifera seed oil has been recognized for its benefits in relation to the skin. The objective of this study was to evaluate the chemical composition and antioxidant activity of moringa seed oil, to formulate a moringa seed oil cream, and to determine the efficacy of moringa seed oil cream in vivo. The chemical components of moringa seed oil were analyzed by high-performance liquid chromatography and gas chromatography. The antioxidant activity of the oil was determined by a 2,2-diphenyl-1-picrylhydrazyl (DPPH) free-radical scavenging assay. An oil-in-water cream containing moringa seed oil was developed and characterized for antioxidant activity. The moringa seed oil cream was further subjected to the accelerated stability test of heating–cooling cycles for six cycles and stored isothermally at 4, 30, and 45 °C for 28 days. The efficacy of moringa seed oil cream was investigated in 32 participants by measuring their skin hydration, erythema, melanin values, and visco-elasticity. The results showed that moringa seed oil contained α-tocopherol, plant sterols, and fatty acids. The oil had antioxidant activity with a 50% of initial concentration (IC50) value of 121.9 mg/mL. The stability study indicated that the pH, viscosity, and rheological behavior of the cream containing moringa seed oil were not significantly changed after storage at 4, 30, and 45 °C for 28 days and six heating–cooling cycles. The moringa seed oil cream exhibited in vitro antioxidant activity and increased the in vivo skin hydration level compared with the cream base. There was no report of skin irritation from moringa seed oil cream application, suggesting that the moringa seed oil cream developed in this study was appropriate for pharmaceutical and cosmetic uses. A M. oleifera seed oil cream was successfully developed. The moringa seed oil cream possessed antioxidant activity, enhanced the skin hydration level, and reduced skin erythema, but did not affect the melanin content and skin visco-elasticity. The moringa seed oil cream did not induce skin irritation and, thus, was safe to use.