Clostridium butyricum (CB) and Phellinus igniarius (PI) have anti-inflammatory, immune regulation, anti-tumor, and other functions. This study aimed to explore the therapeutic effect of CB and mycelium of PI (MPI) alone and in combination on colitis mice induced by dextran sodium sulfate (DSS). Mice were randomly assigned to five groups: (1) control (CTRL), (2) DSS, (3) CB, (4) MPI, and (5) CB + MPI (CON). The weight of the mice was recorded daily during the experiment, and the length of the colon was measured on the last day of the experiment. The colons were collected for hematoxylin and eosin staining, colon contents were collected for intestinal flora analysis, and serum was collected for metabolite analysis. The results showed that compared with the DSS group, CB, MPI, and CON treatments inhibited the weight loss and colon length shortening caused by DSS, significantly increased the concentrations of interleukin (IL)-4, IL-10, and superoxide dismutase, and significantly decreased the concentrations of IL-6, tumor necrosis factor-α, and myeloperoxidase. Gene sequence analysis of 16S rRNA showed that CB, MPI, and CON treatments changed the composition and structure of intestinal microorganisms. Metabolome results showed that CB, MPI, and CON treatments changed serum metabolites in DSS-treated mice, including dodecenoylcarnitine, L-urobilinogen, and citric acid. In conclusion, CB, MPI, and CON treatments alleviated DSS-induced colitis in mice by regulating intestinal flora and metabolites, with the CON group having the best effect.