Pasteurization and freezing are processes used in human milk banks that can affect some of the nutritional and biological properties of human milk. The aim of this study was to evaluate impact of the processing steps adopted by human milk banks on the contents of total phenolic compounds and antioxidant capacity in vitro. Experimental study was realized, in which 40 mL human milk were collected from 8 mothers registered at the Human Milk Bank of Ouro Preto, state of Minas Gerais. Samples were homogenized to form the pool. Human milk was divided into four portions that characterized the treatments: 1) immediately after milking; 2) pasteurization soon after the milking; 3) 7 days after freezing at -8.5 (± 2.8°C) and pasteurized; 4) 14 days after freezing at -8.5 (± 2.8°C) and pasteurized. The content of total phenolic compounds was determined, and the antioxidant capacity was assessed using the 2,2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) and 2,2-diphenyl-1-picrylhydrazyl free radicals scavenging. The results were expressed as mean and standard deviation of six replications. There was a positive correlation between the evaluated methods and reduction in total phenolic content and total antioxidant capacity with slow pasteurization and according to freezing time. There was a negative effect of pasteurization and freezing on the total phenolic content and total antioxidant capacity, and the most significant reduction was observed within the first 7 days of storage.