Clinical antibiotics used worldwide could diminish the intestinal barrier, enhance contact with microbiota and intestinal immune cells, and induce inflammation. We found that ciprofloxacin treatment of Salmonella enterica serovar Typhimurium infection resulted in the destruction of the intestinal barrier, with decreased concentrations of MUC2, ZO-1, and occludin in the jejunum and colon. Ganoderma lucidum ethanol extracts (GLE), as a prebiotic food extract, significantly decreased inflammation-related enzymes, including COX-2, MPO, and iNOS, and pro-inflammatory cytokines (IL-6, IL-1β, IL-17, and TNF-α), and protected the intestinal barrier by increasing the concentration of MUC2, ZO-1, and occludin. Meanwhile it significantly increased the abundances of Salmonella, Parabacteroides, Acinetobacter, Enterococcus, and Escherichia-Shigella, which increased the risk of pathogenic bacterial infections. Prebiotic G. lucidum polysaccharide (GLP) provided a significant intestinal barrier, improving the concentration of ZO-1, occludin, and MUC2 in the colon and jejunum. The synergistic effects of GLP and ciprofloxacin were hypothesized to reverse the negative effects resulting from ciprofloxacin alone, as the concentrations of ZO-1, occludin, and MUC2 were significantly increased in the jejunum and colon, especially in the colon. Also, the synergistic effect increased the abundances of probiotic bacteria Lachnospiraceae NK4A136, Ruminococcaceae UGG-014, Lactobacillus, and Parabacteroides. In conclusion, combined GLP and ciprofloxacin therapy against Salmonella infection alleviated the side effects resulting from the clinical application of the antibiotic alone, and increased the probiotic bacterial population.