In order to investigate the optimum technology of microwave-assisted extraction of polysaccharide from Fortunella margarita (Lour.) Swingle and its antibacterial abilities, the effects of single factors on the yield of polysaccharide were evaluated in this paper. On the basis of these single factor experiments, the extraction technology was optimized by response surface methodology, and then its antibacterial effect was studied. The results showed that the optimum technology conditions were as follows: microwave power of 460 W, microwave time of 8.4 min, ratio of water volume to raw material weight (W/M ratio) of 38 mL/g and particle size of 60 mesh sieve. Under these conditions, the yield of polysaccharides was （5.55±0.13）%, consistent with the theoretical value. Comparing with the hot water and ultrasonic-assisted extraction method, it increased by 207.20% and 33.73%, respectively. Furthermore, the minimal inhibitory concentrations of polysaccharide against Staphyloccocus aureus Rosenbach, Salmonella, Escherichia coli, Bacillus subtilis, and Pseudomonas were 3.13, 50.00, 12.50, 12.50 and 12.50 mg/mL, respectively. Microwave-assisted extraction was an attractive method for extracting polysaccharide from Fortunella margarita (Lour.) Swingle with high efficiency and the polysaccharide displayed a good antibacterial effect on Staphyloccocus aureus Rosenbach.