Autofocusing, as a simple and safe technique, was used to fractionate casein hydrolysate based on the amphoteric nature of its peptides. The antibacterial activity of casein hydrolysate and its autofocusing fractions (A1–10) was examined against Escherichia coli and Bacillus subtilis. The basic fraction A9 exhibited the highest activity with minimum inhibitory concentration (MIC) of 150 μg/mL, whereas casein hydrolysate showed MIC values ranging from 2000 to 8000 μg/mL. The antibacterial peptides in A9 were purified by using a series of size exclusion and reversed phase chromatographies. Three peptides exhibited the most potent antibacterial activity with MIC values ranging from 12.5 to 100 μg/mL. These peptides were generated from αs2-casein, αs1-casein, and κ-casein and identified as K165KISQRYQKFALPQYLKTVYQHQK188, I6KHQGLPQEV15, and T136EAVESTVATL146, respectively. Therefore, the results revealed that casein hydrolysate had potent antibacterial peptides that could be isolated by autofocusing technique. © 2012 International Union of Biochemistry and Molecular Biology, Inc.