Thermophilic bacteria and spores can survive pasteurization temperatures and reduce shelf life of milk. These bacteria produce enzymes that produce free fatty acids and peptides altering the quality of milk. Coupling ultrasound with pasteurization may aid in reducing the surviving microbes in fluid foods and enhancing the product quality and shelf life based on previous studies.This study determined the D-value of Geobacillus stearothermophilus (GS) and Anoxybacillus flavithermus (AF) in 2% fat milk and tryptic soy broth (TSB) medium when treated with thermosonication in a batch system. Sterile 2% fat milk and TSB were inoculated with GS and AF and aliquots were plated on TSA plates before and after control and thermosonication treatments for enumeration to calculate the D-values. Batch sonication was performed using QSonicaQ500 (Newtown, CT) using a 10 ml flow cell and a 3.2 mm sonication microtip at 72 and 73°C and treated for 5 to 120 s (5 mL sample volume). D-values for GS in TSB and 2% milk with heat only at 72°C were >60 s and at 73°C were 60.02 and 63.92 s, respectively. With thermosonication, the D-values in TSB were 25.16 and 26.49 s at 72 and 73°C. D-values for GS in milk at 72 and 73°C with thermosonication were 19.98 and 18.68 s, respectively. Similarly, for AF, D-values in TSB and 2% milk with heat only at 72°C were >60 s and at 73°C were 48.41 and 56.11 s, respectively. Thermosonication decreased the D-values of AF in TSB to 44.39 s at 72°C and 36.91 s at 73°C and in 2% milk to 38.54 s at 72°C and 35.11 s at 73°C. ANOVA revealed that thermosonication had a statistically significant effect on GS reduction in TSB and milk but temperature did not. In the case of AF, although an increase in temperature and application of thermosonication decreased the D-values, ANOVA revealed that neither temperature nor thermosonication had a significant effect on the reduction in D-values. Studies are being performed to see the effect of thermosonication on D-values of GS and AF spores. Treating milk with pasteurization along with thermosonication may potentially reduce the microbial load of thermophilic organisms and spores in milk leading to products with extended shelf lives.