The inactivation of Bacillus subtilis spores by ultrasonic treatments under static pressure (Mano‐Sonication, MS) and a combined MS/heat treatment (Mano‐Thermo‐Sonication) was investigated. The sporicidal effect of MS treatments depended on static pressure, amplitude of ultrasonic waves and treatment temperature. At 70 °C, pressure increments up to 500 kPa caused progressively more inactivation. An MS treatment at 500 kPa and 117 μm of amplitude for 12 min inactivated approximately 99% of the B. subtilis spore population. Over 500 kPa, further increments in pressure did not increase the percentage of inactivation. In the range 90–150 μm, an exponential relationship was observed between the amplitude of ultrasonic waves under pressure and the number of survivors. While an MS treatment (20 kHz, 300 kPa, 70 °C, 12 min) at 90 μm inactivated 75% of the B. subtilis spore population, the same treatment at 150 μm inactivated 99·9% of this population. The MS treatments at temperatures higher than 70 °C (MTS) led to more spore inactivation. In the range 70–90 °C, the combination of heat with an MS treatment (20 kHz, 300 kPa, 117 μm, 6 min) had a synergistic effect on spore inactivation. The inactivating effect of ultrasound was due neither to titanium particles eroded from the sonication tip, nor to free radicals released during ultrasonic treatment. The MS treatments sensitized spores of B. subtilis to lysozyme.