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The 2015-2020 dietary guidelines for Americans advise substituting total unsaturated fatty acids (Sigma UFA) for total saturated fatty acids (Sigma SFA). Thus, field experiments were carried out to verify the influence of irrigation regime (well-irrigated and water deficit) and fertilizers (chemical and biological) on the ratio of Sigma UFA to Sigma SFA of evening primrose seed oil. Therefore, two experiments were conducted at the experimental stations (arid and semi-arid) of Iran in 2014 and 2015. Experiments were conducted in a split factorial layout within a randomized complete block design with three replications. Water deficit significantly reduced UFA (omega 3, 6, 7 and 9), Sigma UFA and ratio of Sigma UFA to Sigma SFA (especially in the arid region), but it increased SFA and ESFA (especially in the arid region). In fact, fatty acid quality (increased ratio of Sigma UFA to Sigma SFA) of evening primrose seed oil was significantly increased in well-irrigated compared to water deficit stress (especially in the semi-arid region). Bio-fertilizers (Azospirillum lipoferwn and Glomus mosseae) and chemical fertilizers (urea + triple superphosphate) increased the ratio of Sigma UFA to Sigma SFA of evening primrose seed oil (especially in the semi-arid region), but fatty acid quality of evening primrose oil was significantly increased in bio-fertilizers compared to the chemical fertilizers (especially in the arid region).