Background: Studies have shown that omega-3 (n-3) Polyunsaturated Fatty Acids (PUFAs), including docosahexaenoic acid (DHA), might have beneficial effects on somatic and mental health, potentially partly due to their mitigating effects on three major biological stress systems: the immune-inflammatory system, the hypothalamic-pituitary-adrenal-axis (HPA-axis) and the autonomic nervous system (ANS). Objective: To examine the association between (cumulative measures of) markers of three biological stress systems and n-3 PUFA and DHA plasma levels. Design: Plasma n-3 PUFA and DHA were measured using Nuclear Magnetic Resonance in 2724 participants from the Netherlands Study of Depression and Anxiety. Linear regression analyses (adjusted for sociodemographic, sampling, lifestyle and somatic disease variables) associated inflammation (C-reactive protein, interleukin-6, tumor necrosis factor alpha), HPA-axis (cortisol awakening response and evening cortisol) and ANS (heart rate, respiratory sinus arrhythmia and pre-ejection period) markers and cumulative indices within and across stress systems as independent variables with n-3 PUFA and DHA levels as dependent variables. Results: Participants had a mean age of 41.8 (SD = 13.1) and 65.7% were female. Higher levels of all three inflammation markers (Beta=−.146 to −.073, all p-values<.001), evening cortisol (Beta=−.045, p =.033) and heart rate (Beta=−.080, p < 0.001) were significantly negatively associated with n-3 PUFA. Suggesting an exposure-response relationship, a higher number of markers indicative of inflammation and hyperactive HPA-axis (p <.001 and p =.003, respectively), but not of ANS dysregulation, was found in persons with lower n-3 PUFA levels. An exposure-response relationship was also found for having a higher number of different stress system dysregulations with lower n-3 PUFA levels (p <.001). For DHA, results were in line with those for n-3 PUFA, although with slightly smaller effect sizes. Conclusions: Our study confirmed that having various (cumulative) indicators of dysregulation of three biological stress systems was significantly associated with lower n-3 PUFA and DHA plasma levels. If low n-3 PUFA levels are the cause of dysregulated stress systems, then n-3 PUFA supplementation might reduce biological stress and thereby improve somatic and mental health.