Manothermosonication (MTS) has been one of the key alternatives to current pasteurization techniques. This study evaluates the effectiveness of the MTS system in the inactivation of two opposite gram-stained bacteria: E. cola ATCC 25922 and S. aureus ATCC 25923. Samples were treated with different conditions, including mild heat (T, 50 degrees C, 100 kPa), sonication (US, 30 degrees C, 100 kPa), thermosonication (TS, 50 degrees C, 100 kPa), manosonication (MS, 30 degrees C, 400 kPa), and manothermosonication (MTS, 50 degrees C, 400 kPa) in pulse mode for precise temperature control. After 5 min, the viability of E. coli. and S. aureus treated with MTS decreased by 6.25 and 4.55 log CFU/mL, and the maximum decrease in non-linear Biphasic decimal reduction (D-value) of the sensitive population was 91.35% and 94.24%, respectively. Since pressurized ultrasound generated excess heat of up to 0.88 degrees C/sec, an inactivation experiment employing such heat resulted in minimal linear D-values of 0.17 (E. coli) min and 0.18 min (S. aureus) with power intensity of 3.69 Watt/mL thermally or 14.29 Watt/mL electrically. All experiments indicated that gram-positive S. aureus was more resistant to sonication than gram-negative E. coli, but the lethality rate became more comparable with more violent cavitation.