To investigate the effects of different components of borneol essential oil, separated by molecular distillation, on the motility and biofilm formation of Escherichia coli, the study was conducted as follows: First, gas chromatography-mass spectrometry (GC-MS) was employed to analyze the volatile components of the crude borneol essential oil, its light fraction, and its heavy fraction. Next, the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the three components against E. coli ATCC 25922 were determined using the double dilution method and plate coating method, respectively. Finally, the impact of these three components on the swimming and swarming motility, as well as biofilm formation of E. coli ATCC 25922, was studied using motility assays and crystal violet staining. The results show that the crude borneol essential oil, light fraction, and heavy fraction contain 27, 24, and 19 volatile components, respectively. The highest relative content of volatile components is D-borneol (19. 93%) in the crude oil, eucalyptol (19.36%) in the light fraction, and D-borneol (28.92%) in the heavy fraction.The MIC and MBC of the crude oil, light fraction, and heavy fraction against E. coli are 6.25 µL/mL, and all three components are able to delay bacterial growth at sub-inhibitory concentrations. At 1/2 MIC and 1/4 MIC, all three borneol essential oil components reduce the swimming and swarming motility of E. coli and inhibited biofilm formation. The heavy fraction of borneol essential oil exhibits superior inhibition of E. coli motility and biofilm formation compared to the crude oil and light fraction. These findings indicate that molecular distillation can enrich the antibacterial active components in borneol essential oil, effectively enhancing its added value and demonstrating broad application prospects.