Stress related to parenting a child with autism spectrum disorder can differently affect caregiver's physiological reactivity to acute stress. Here, parental stress levels, psychological characteristics, and coping strategies were assessed alongside measures of heart rate, heart rate variability, and cortisol during a psychosocial stress test in mothers of children with ASD (M-ASD, n = 15) and mothers of typically developing children (n = 15). M-ASD reported significantly higher levels of parental stress, anxiety, negative affectivity, social inhibition, and a larger preference for avoidance strategies. M-ASD showed larger heart rate and cortisol responses to the psychosocial stress test. A positive relationship was found between parental stress levels and the magnitude of the cortisol stress response in both groups. The present findings indicate exaggerated physiological reactivity to acute psychosocial stress in M-ASD and prompt further research to explore the role of individual differences in mediating the effects of parental stress on physiological stress responses.