Hyperbaric oxygenation (HBO) treatment protocols utilize low pressures up to 3ATA. Higher pressures may induce side effects such as convulsions due to brain toxicity. The optimal HBO pressure allowing for maximal therapy and minimal toxicity is under controversy. However, it can be evaluated by monitoring oxygen delivery, saturation, and consumption. In this study, the monitoring system fixed on the rats’ brain cortex included a time-sharing fluorometer-reflectometer for monitoring mitochondrial NADH and hemoglobin oxygenation (HbO₂) combined with Laser Doppler Flowmetry (LDF) for blood-flow monitoring. Rats were located in a hyperbaric chamber and exposed to different pressures. The HBO pressure caused an increase in HbO₂ and a decrease in NADH in proportion to the increase in hyperbaric pressure, up to a nearly maximum effect at 2.5ATA. At 6ATA, 15 minutes before convulsions started, blood volume and NADH started to increase, while tissue O₂ supply by hemoglobin remained stable. Oxygen pool includes oxygen dissolved in the plasma and also bounded to hemoglobin. Above 2.5ATA, hemoglobin is fully saturated and the oxygen pool nourishment derives only from the oxygen dissolved in the plasma, exceeding the physiological ability for autoregulation; hence, homeostasis is disturbed and convulsions appear. This information is vital because pressures around 2.5ATA–3ATA are standard clinically applied pressures used to treat most of the pathophysiological problems considering the potential benefit which must be balanced against the potential toxicity. This study enables, for the first time, to evaluate the oxygenation level of hemoglobin in the microcirculation. Furthermore, our study showed that additional oxygen pressure (above 2.5ATA) caused brain oxygen toxicity within a short variable period of time after the pressure elevation.