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Ultrasonic technology is being increasingly explored in minerals processing. In this paper, ultrasonic treatment was introduced as a novel method for microbial desulfurization of sulfide ores. A Box–Behnken experiment was performed to find the best combination of factor levels for the following experiments; consequently, the combination of factor levels at the maximum 5-day average desulfurization rate at 20 g of ore was a particle size of 120–140 mesh, a shaker speed of 175 rpm, and a dosage of 111 mL of bacterial solution. Under these conditions, a microbial desulfurization experiment of sulfide ores by ultrasonic treatment was carried out, and the effect of the particle size, the ultrasonic action time, and the ultrasonic power were investigated. Results indicated that the ultrasonic effect was not significant for ore samples with larger particle sizes, and the appropriate increase in ultrasonic action time was beneficial to the improvement of desulfurization rate, but the effect showed a decreasing trend when it exceeded 50 min, and the best desulfurization effect was achieved when the power was 300 W. This study demonstrated that the effect of microbial desulfurization can be greatly enhanced by ultrasonic treatment.