Pumpkin seed oil is a by-product, abundant in nutrients and bioactive components that promote several health benefits. This study aimed to compare chemical compositions, antioxidant, and pharmacological activities of pumpkin seed oils extracted from Cucurbita moschata Duch. Ex Poir. (PSO1) and Cucurbita moschata (Japanese pumpkin) (PSO2) by aqueous enzymatic extraction. An enzyme mixture consisting of pectinase, cellulase, and protease (1:1:1) was used in the enzymatic extraction process. Fatty acid composition of the oils was determined using fatty acid methyl ester/gas chromatographic-mass spectrometry. Antioxidant activity assays were measured by using stable free radical diphenylpicrylhydrazyl, radical cation 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate, ferric reducing/antioxidant power, and ferric thiocyanate assay. Inhibition of enzymes involving skin aging and whitening process was investigated. Linoleic acid was a major component of all pumpkin seed oils. Additionally, there was also a significant amount of oleic acid, palmitic acid, and stearic acid detected. PSO2 possessed the highest antioxidant activities compared to PSO1 and commercial pumpkin seed oils (COM1 and COM2). Both PSO1 and PSO2 exhibited higher inhibitory effects on hyaluronidase, collagenase, and tyrosinase than the commercials. Therefore, aqueous enzymatic extraction could yield pumpkin seed oils with higher antioxidant, anti-aging, and whitening activities. This is beneficial for further pharmacological studies and can be used as a functional food for skin benefits.