Human blood contains cell-free mitochondrial DNA (cf-mtDNA) that dynamically increases in concentration in response to acute mental stress. Like other neuroendocrine stress markers, we previously found that cf-mtDNA is also detectable in saliva, calling for studies examining saliva cf-mtDNA reactivity to mental stress. In the present study, participants from the MiSBIE (Mitochondrial Stress, Brain Imaging, and Epigenetics) study (n = 68, 66 % women), were exposed to a brief socio-evaluative stressor, which induced a striking 280 % or 2.8-fold increase in saliva cf-mtDNA concentration within 10 min (g=0.55, p < 0.0001). In blood drawn concurrently with saliva sampling, stress increased cf-mtDNA by an average 32 % at 60 min in serum (g=0.20), but not in anticoagulated plasma where cf-mtDNA decreased by 19 % at 60 min (g=0.25). Examining the influence of mitochondrial health on cf-mtDNA reactivity in participants with rare mitochondrial diseases (MitoD), we report that a subset of MitoD participants exhibit markedly blunted saliva cf-mtDNA stress reactivity, suggesting that bioenergetic defects within mitochondria may influence the magnitude of saliva, and possibly blood cf-mtDNA responses. Our results document robust saliva cf-mtDNA stress reactivity and provide a methodology to examine the psychobiological regulation of cell-free mitochondria in future studies.