Increases in fractional exhaled nitric oxide (FeNO) have been observed after acute laboratory stress, which could indicate a strengthening of immune defenses in acute stress because of the quick onset of the response and the role of nitric oxide in airway-protective functions. In addition, because sustained psychological distress and depression are known to deteriorate immune defenses systems, they may dampen the FeNO response to acute stress. FeNO and negative affect were measured before and after a speech and mental arithmetic stressor. We examined the association of stress-induced FeNO changes with momentary negative affect and questionnaires of perceived stress, anxious mood, and depressive mood in 39 asthma patients and 41 healthy controls. FeNO increased from baseline to stress in participants with asthma (from 3.38 [0.102] to 3.46 [0.103] ln(ppb)) and controls (2.86 [0.098] to 2.92 [0.099]; F(4,141) = 3.26, p = .014), but the magnitude of the FeNO response did not differ between groups (F < 1). Only low levels of depressive mood were associated with FeNO increases after stress (most pronounced at 0 minute poststress; t(76) = 3.87, p < .001). In contrast, only higher perceived stress was associated with FeNO increases (most pronounced at 0 minute poststress; t(75) = 4.09, p < .001), and momentary negative affect was associated with higher FeNO throughout assessments (β = 0.08, t(114) = 8.27, p = .005). Associations of FeNO with psychological variables were largely unrelated to asthma status and inhaled corticosteroid use. Depressive mood is associated with a reduced mobilization of airway nitric oxide in acute stress, whereas other indicators of negative affect are positively associated with overall FeNO levels and reactivity.