Background: Childhood trauma (CT) is a risk factor for depressive and anxiety disorders. Although dysregulated biological stress systems may underlie the enduring effect of CT, the relation between CT and separate and cumulative activity of the major stress systems, namely, the hypothalamic-pituitary-adrenal (HPA)-axis, the immune-inflammatory system, and the autonomic nervous system (ANS), remains inconclusive. Methods: In the Netherlands Study of Depression and Anxiety (NESDA, n = 2778), we determined whether self-reported CT (as assessed by the Childhood Trauma Interview) was associated with separate and cumulative markers of the HPA-axis (cortisol awakening response, evening cortisol, dexamethasone suppression test cortisol), the immune-inflammatory system (C-reactive protein, interleukin-6, tumor necrosis factor-α), and the ANS (heart rate, respiratory sinus arrhythmia, pre-ejection period) in adulthood. Results: Almost all individuals with CT (n = 1330) had either current or remitted depressive and/or anxiety disorder (88.6%). Total-sample analyses showed little evidence for CT being significantly associated with the separate or cumulative stress systems’ activity in adulthood. These findings were true for individuals with and without depressive and/or anxiety disorders. To maximize contrast, individuals with severe CT were compared to healthy controls without CT. This yielded slight, but significantly higher levels of cortisol awakening response (AUCg, β =.088, p =.007; AUCi, β =.084, p =.010), cumulative HPA-axis markers (β =.115, p =.001), C-reactive protein (β =.055, p =.032), interleukin-6 (β =.053, p =.038), cumulative inflammation (β =.060, p =.020), and cumulative markers across all systems (β =.125, p =.0003) for those with severe CT, partially explained by higher rates of smoking, body mass index, and chronic diseases. Conclusion: While our findings do not provide conclusive evidence on CT directly dysregulating stress systems, individuals with severe CT showed slight indications of dysregulations, partially explained by an unhealthy lifestyle and poorer health.