Goat whey hydrolysates were produced from goat whey using Alcalase ® from Bacillus licheniformis. The goat whey hydrolysate (GWH) with the highest degree of hydrolysis was fractionated by size exclusion chromatography (SEC), and the antibacterial activity of the GWH and the obtained SEC-fractions were evaluated using the disc diffusion method. The results showed that the GWH exhibited significantly higher antibacterial activity than the unhydrolysed goat whey. Among all SEC fractions obtained, only SEC-F 2 and SEC-F 3 showed antibacterial activity against the tested bacteria. SEC-F 3 exhibited the highest antibacterial effectiveness against Escherichia coli and Bacillus cereus with MIC values of 0.09 and 0.03 mg/mL respectively, whereas, SEC-F 3 and SEC-F 2 had comparable MIC values against Staphylococcus aureus (~0.02 mg/mL). Transmission electron microscopy showed that the GWH caused changes in the bacterial cells affected. Additionally, LCeMS analysis was used to characterize the peptides of GWH and SEC-F 3. Two major peptides were detected in GWH and SEC-F 3 , with higher concentration in the latter, having masses of 730 and 1183 Da, respectively. These results suggest that hydrolysis of goat whey by Alcalase is an easy tool to enhance its antibacterial activities. GWH and its effective antibacterial fractions may be used to control the undesirable bacteria in food and improve its safety.