Diarrhea, a common gastrointestinal symptom in health problems, is highly associated with gut dysbiosis. The purpose of this study is to demonstrate the effect of multistrain probiotics (Sensi-Biome) on diarrhea from the perspective of the microbiome-neuron axis. Sensi-Biome (Lactiplantibacillus plantarum, Bifidobacterium animalis subsp. lactis, Lactobacillus acidophilus, Streptococcus thermophilus, Bifidobacterium bifidum, and Lactococcus lactis) was administered in a 4% acetic acid-induced diarrhea rat model at concentrations of 1 × 10 (G1), 1 × 10 (G2), and 1 × 10 CFU/0.5 mL (G3). Diarrhea-related parameters, inflammation-related cytokines, and stool microbiota analysis by 16S rRNA were evaluated. A targeted and untargeted metabolomics approach was used to analyze the cecum samples using liquid chromatography and orbitrap mass spectrometry. The stool moisture content (p < 0.001), intestinal movement rate (p < 0.05), and pH (p < 0.05) were significantly recovered in G3. Serotonin levels were decreased in the multistrain probiotics groups. The inflammatory cytokines, serotonin, and tryptophan hydroxylase expression were improved in the Sensi-Biome groups. At the phylum level, Sensi-Biome showed the highest relative abundance of Firmicutes. Short-chain fatty acids including butyrate, iso-butyrate, propionate, and iso-valeric acid were significantly modified in the Sensi-Biome groups. Equol and oleamide were significantly improved in the multistrain probiotics groups. In conclusion, Sensi-Biome effectively controls diarrhea by modulating metabolites and the serotonin pathway.