Abstract: To investigate the structural characteristics of water soluble polysaccharides in the pulp of Annona squamosa L (WPAS) and their antibacterial effects against foodborne pathogens, WPAS was extracted using ultrasound assisted extraction, then purified. Its structures were characterized using ion chromatography, high-performance gel permeation chromatography, infrared spectroscopy, Congo red assay, X-ray diffraction , Zeta potential analysis, and scanning electron microscopy. The results indicated that WPAS was a neutral polysaccharide composed mainly of arabinose as the primary monosaccharide, with an average molecular weight of 14.99 kDa, an amorphous structure, a porous network morphology on the surface, and good stability in solution. In vitro antibacterial assays revealed that at a mass concentration of 20 mg/mL, the antibacterial zone diameters against Bacillus subtilis and Pseudomonas aeruginosa were 20.34±0.75 and 10.21±1.47 mm, respectively, and minimum inhibitory concentrations of 12.50 and 17.50 mg/mL, respectively. Antibacterial efficacy against Bacillus subtilis and Pseudomonas aeruginosa was evaluated using cell-membrane permeability assays and adenosine triphosphatase (ATPase) activity measurements. The results showed that WPAS disrupted bacterial cell membrane integrity and significantly inhibited ATPase activity, thereby exerting significant antibacterial effects. In summary, WPAS demonstrates significant antibacterial activity against Bacillus subtilis and Pseudomonas aeruginosa. These findings provide a theoretical foundation for the application of Annona squamosa L polysaccharides in food preservation and functional product development.