Mycobacterium vaccae is a species of nonpathogenic bacterium that lives naturally in soil. This study compared the physiological effects at a metabolomic level with autonomic nervous system responses in adults during soil-mixing activities, based on the presence or absence of M. vaccae in the soil. Twenty-nine adult participants performed soil-mixing activities for 5 minutes using sterilized soil with culture media and M. vaccae, respectively. Blood samples were drawn twice from each participant after each activity. Electroencephalograms and electrocardiograms were measured during the activity. Serum metabolites underwent metabolite profiling by gas chromatography, followed by multivariate analyses. Soil-emitted volatile organic compounds were identified using the solid-phase microextraction and gas chromatography-mass spectroscopy, followed by multivariate analyses. The volatile compound analysis revealed that the metabolites related to esters and sulfur-containing compounds are greater in soil with M. vaccae. Serum metabolomics revealed that the treatment group (soil inoculated by M. vaccae) possesses relatively higher levels of inter-alia organic and amino acids compared with the control group (soil mixed with culture media). In the treatment group, the electroencephalogram and electrocardiogram revealed that alpha band activity of the occipital lobe increases, while heart rate decreases. This study concludes that M. vaccae soil contact can affect human metabolic and autonomic reactions.