Tactical occupations regularly encounter life-threatening situations while on duty. Although these occupations are often trained to utilize slow breathing (SB) during intense stress, there is no evidence supporting the effects on markers of stress in response to a virtual reality active shooter training drill (VR-ASD). The purpose of the study was to determine the impact of acute SB on biomarkers of stress in response to a VR-ASD. Seventy-nine (n = 79) subjects performed either slow breathing method 1 (SB1), slow breathing method 2 (SB2), or normal breathing (control) for five minutes, both pre- and post-VR-ASD. Saliva samples were analyzed for stress markers, including α-amylase (sAA) and secretory immunoglobulin-A (SIgA). Both methods of SB resulted in significantly lower sAA concentrations at 5 (p < 0.001) and 30 min post-VR-ASD (SB1: p = 0.008; SB2: p < 0.001) compared to the control. In the control condition, the sAA concentrations were significantly elevated 5 min post-VR-ASD (p < 0.001) but did not change across time in SB1 or SB2 (p > 0.05). Thus, both SB1 and SB2 reduced the sAA response and resulted in lower concentrations post-VR-ASD. This study was pre-registered as a clinical trial (“Impact of Breathing Interventions on Stress Markers”; NCT05825846).