This study aimed to evaluate the effect of thermosonication of human milk (HM) followed by frozen storage on its lipid stability, antioxidant activity, and tocopherol content. The treatments investigated in this study corresponded to a factorial structure with additional treatment (control), having processing method and storage time as factors. The additional treatment corresponds to raw milk, with 0 days of storage. The experimental design was the one randomly randomized with three replications. Essential fatty acids and antioxidant activity were conserved by the ferric reducing antioxidant power method but decreased by the 2,2‐diphenyl‐1‐picrylhydrazyl method (9%) in both pasteurized and thermosonicated samples. Only tocopherols showed greater degradation with thermosonication. In freezing, regardless of processing, the same behavior was observed for the analyzed parameters, with a reduction in antioxidant activity and tocopherol isoforms. Hexanal increased during storage for up to 30 days (0.87 μL/mL of HM) regardless of processing. Thermosonication of HM proved to be a method similar to slow pasteurization in the studied parameters. It is suggested that further studies should be conducted on the use of ultrasound as an alternative processing technique to the conventional processes adopted by the Human Milk Bank. Practical Applications Thermosonication proved to be a method similar to pasteurization in the studied parameters. This study is extremely relevant to assist in the determination of new processing techniques to obtain donated HM with better nutritional quality. It is suggested that further studies should be conducted on the use of ultrasound as an alternative processing technique to the conventional processes adopted by the Human Milk Bank.