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In this paper, a novel technique to achieve precise temperatures in food sterilization has been proposed. An accurate temperature profile is needed in order to reach a commitment between the total removal of pathogens inside the product and the preservation of nutritional and organoleptic characteristics. The minimal variation of the target temperature in the sample by means of a monitoring and control software platform, allowing temperature stabilization over 100 °C, is the main goal of this work. A cylindrical microwave oven, under pressure conditions and continuous control of the microwave supply power as function of the final temperature inside the sample, has been designed and developed with conditions of single-mode resonance. The uniform heating in the product is achieved by means of sample movement and the self-regulated power control using the measured temperature. Finally, for testing the sterilization of food with this technology, specific biological validation based on Bacillus cereus as a biosensor of heat inactivation has been incorporated as a distribution along the sample in the experimental process to measure the colony-forming units (CFUs) for different food samples (laboratory medium, soup, or fish-based animal by-products). The obtained results allow the validation of this new technology for food sterilization with precise control of the microwave system to ensure the uniform elimination of pathogens using high temperatures.