Lactobacillus spp. and Bifidobacterium spp. was used to protect against gastrointestinal disorders. The present study evaluated the effects of probiotic mixture (PM) containing Lactobacillus spp. and Bifidobacterium spp. on intestinal mucositis induced by 5-fluorouracil (5-FU). Swiss male mice (25-30 g) were treated with 5-FU (450 mg/kg, ip) and were orally administered (PM). Probiotic mixture 1 (PM-1) is a mixture of two probiotics (Lactobacillus acidophilus and Bifidobacterium lactis) and probiotic mixture 2 (PM-2) is a mixture of four probiotics (Lactobacillus acidophilus, Lactobacillus paracasei, Lactobacillus rhamnosus, and Bifidobacterium lactis). PM-1 and PM-2 decreased histopathological scores in the duodenum and jejunum after mucositis. PM-2 attenuated 5-FU-induced weight loss. On the other hand, PM-1 did not exert a significant effect on weight loss. Both probiotics mixture increased the villus/crypt ratio in all intestinal segments, increased GSH levels in the duodenum and jejunum, and reduced the MDA, MPO, TNF-α, and IL-6 levels in the duodenum, jejunum, and ileum. PM-2 attenuated the delay in gastric emptying. PM-1 and PM-2 prevented epithelial injury in intestinal mucositis by 5-FU, demonstrating the potential use of these probiotics as therapeutic agents against intestinal mucositis.