In order to better understand stress responses, neuroimaging studies have investigated the underlying neural correlates of stress. Amongst other brain regions, they highlight the involvement of the prefrontal cortex. The aim of the present study was to explore haemodynamic changes in the prefrontal cortex during the Maastricht Acute Stress Test (MAST) using mobile functional Near-Infrared Spectroscopy (fNIRS), examining the stress response in an ecological environment. The MAST includes a challenging mental arithmic task and a physically stressful ice-water task. In a between-subject design, participants either performed the MAST or a non-stress control condition. FNIRS data were recorded throughout the test. Additionally, subjective stress ratings, heart rate and salivary cortisol were evaluated, confirming a successful stress induction. The fNIRS data indicated significantly increased neural activity of brain regions of the dorsolateral prefrontal cortex (dlPFC) and the orbitofrontal cortex (OFC) in response to the MAST, compared to the control condition. Furthermore, the mental arithmetic task indicated an increase in neural activity in brain regions of the dlPFC and OFC; whereas the physically stressful hand immersion task indicated a lateral decrease of neural activity in the left dlPFC. The study highlights the potential use of mobile fNIRS in clinical and applied (stress) research.