The neurovisceral integration model proposes that information flows bidirectionally between the brain and the heart via the vagus nerves and vagally-mediated heart rate variability (vmHRV) can be used to index heart-brain interaction. Recent research has shown that voluntary reduction of breathing rate (slow-paced breathing, SPB) can enhance cardiac vagal control. Additionally, prefrontal transcranial direct current stimulation (tDCS) can modulate the excitability of the prefrontal region and influence the vagus nerve. However, fundamental research on the combination of SPB and prefrontal tDCS to increase vmHRV and other physiological indices of the autonomic nervous system is scarce. Therefore, 200 healthy participants were assigned to four experimental groups. Each group received either 20 min of active or sham tDCS combined with 5.5 breath per minute (BPM) or 15 BPM breathing. Regardless of the tDCS condition, the SPB group showed a significant increase in vmHRV over 20 minutes, suggesting an increase in parasympathetic activity. In addition, a significant decrease in HR at the first and second 5-minute epochs of the intervention. Regardless of breathing condition, the active tDCS group exhibited higher HR at the fourth 5-minute epoch of the intervention compared to the sham tDCS group, suggesting more sympathetic arousal. However, there was no combined effect on vmHRV, HR, skin conductance, or blood pressure. SPB is a robust technique for increasing vmHRV, whereas prefrontal tDCS may produce effects that counteract those of SPB. More research is necessary to test whether and how top-down and bottom-up approaches can be combined to improve vagal control.