In this study, we explored the fractionation and identification of antibacterial peptides from cottonseed protein hydrolysates obtained using Alcalase. The cottonseed protein hydrolysates were preliminarily separated using Sephadex C-25, and fraction P5 exhibited antibacterial activity against four tested strains, including Gram-positive and Gram-negative bacteria, at concentrations ranging from 1 to 4 mg/mL. The potential antibacterial peptides in fraction P5 were then isolated by reverse-phase high-performance liquid chromatography based on a specific reaction of Arg with benzaldehyde. Nine novel peptides encoded in cottonseed proteins were identified by mass spectrometry, and three peptides (KDFPGRR, LGLRSGIILCNV, and DENFRKF) with antibacterial activities of 77.7%, 69.3%, and 45.0% at 1.0 mg/mL, respectively, were chemically synthesized. Fluorescence labeling and staining assays confirmed that antibacterial peptides could combine and interact with the surface of the cell membrane. In addition, sensitivity analysis of the environmental conditions revealed that antibacterial peptides were not influenced by temperature and pH, but they were sensitive to Ca2+, with a decrease in antibacterial activity decreased of 40% occurring as the concentration of Ca2+ increased from 0.05% to 5%.Our results indicated that cottonseed protein could be used as a potential source of antibacterial peptides that could be applied to food systems and the feed industry. Display omitted •Antimicrobial peptides were separated by specific reaction of arginine.•Nine potential antibacterial bioactive peptides were identified.•The peptide KDFPGRR showed the highest antibacterial activity.•Antibacterial peptides destroy the cells by interaction with cell membrane.