Cucumber (Cucumis sativus) seed oil has the potential for use as an edible oil and as a pharmaceutical, cosmetic, insecticidal, and industrial product. In this study, we investigated, for the first time, the effect of cultivar and season on seed number, oil content, and fatty acid profiles as well as their proportions in different cucumber cultivars. We examined the effects of spring and autumn seasons on seed oil content and fatty acid composition in 46 cucumber cultivars and one wild species of cucumber (C. anguria) grown in greenhouse experiments in 2013 and 2014. Seed oil was determined using the Soxhlet method and fatty acids using the gas chromatography-mass spectrometry method. Seed oil content in the cucumber seeds ranged from 41.07% in ‘Hazerd’ to 29.24% in ‘Lubao’ while C. anguria had 23.3%. Fatty acids detected were linoleic (C18:2), palmitic (C16:0), oleic (C18:1), stearic (C18:0), linolenic (C18:3), behenic (C22:0), arachidic C20:0), lignoceric (C24:0), eicosenoic (C20:1), palmitoleic (C16:1), and myristic (C14:0), among other unidentified fatty acids. The results showed significant effects of cultivar genotype, growing season, and interactions on the variables examined. The content of seed oil and fatty acids differed significantly among the cultivar genotypes. Spring-grown cucumbers had higher quantities of oil than the autumn-grown cucumbers. The content of fatty acids (mainly palmitic, palmitoleic, stearic, oleic, eicosenoic, and lignoceric) also was higher in spring. In autumn there were more seeds, and higher linoleic, linolenic, and other unspecified fatty acids. The higher the oleic acid content the lower was the linoleic acid indicating a strong negative relationship in these two fatty acids. The higher the seed oil content the higher was linoleic and oleic indicating a positive relationship between the seed oil and the two fatty acids. Results of this study provide important information applicable in improving management and production of cucumber seed oil especially considering its versatility in uses. Furthermore, the wide range of fatty acids found in the studied cucumber cultivars could be used in the production of novel industrial oils through genetic engineering.