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To improve the stability of grapefruit seed extract (GSE) and prolong its antibacterial effect, a GSE nanoemulsion (GNE) was prepared by a high‐energy emulsifying method. The particle size and PDI of fresh GNE were 173.9 nm and 0.105, respectively. TEM images showed that uniform and closely distributed GNE droplets. The minimum inhibitory concentration (MIC) of GNE against Staphylococcus aureus and Escherichia coli were 0.6 wt% and 1.2 wt%, respectively, 25% and 14% improved compared to GSE. Besides, the GNE was proved to be more effective to destroy the cell wall of E. coli than that of S. aureus by measuring the concentration of alkaline phosphatase after treatment. The increase of conductivity, nucleic acid, and protein released from the cells indicated that the bacteria cell membrane has been severely damaged after GNE treatment. TEM results further illustrated that the internal microstructure of bacteria cells was significantly changed with GNE treatment. Novelty impact statement: In the present study, the grapefruit seed extract nanoemulsion (GNE) with more surface‐volume ratio, higher biological activity, and stronger bioavailability was successfully prepared using the high energy method. GNE has an inhibitory effect on S. aureus and E. coli by destroying the cell wall and cell membrane of the bacteria. This work provides support for expanding the application of grapefruit seed extract. [ABSTRACT FROM AUTHOR]