The development of ecologically based in vivo microecological formulations for treating Clostridioides difficile infection (CDI) is a current research focus. Here, we selected three microorganisms-Akkermansia muciniphila (AM), Bacteroides ovatus (BO), and Bifidobacterium breve (BB)-to formulate a mixed bacterial formulation (ABB). Subsequently, we evaluated the ecological interactions among these three microorganisms and investigated their therapeutic efficacy in a CDI murine model. Our investigation revealed the presence of a commensalism relationship among AM, BO, and BB. These microorganisms collectively formed a robust and densely packed symbiotic biofilm, with BB being the predominant member in terms of numerical abundance. This phenomenon was concomitant with a marked elevation in the levels of AI-2 and c-di-GMP. ABB exhibits the capability to inhibit crucial biological indicators of C. difficile (CD), such as toxin production, through the secretion of substantial quantities of lactic acid. Additionally, ABB indirectly suppresses CD by activating the NF-κB signaling pathway in Raw 264.7 cells, which stimulates the secretion of significant quantities of IL-6, IL-8, TNF-α, and IL-1β. ABB demonstrated exceptional efficacy in a CDI murine model, as evidenced by a substantial enhancement in survival rates and intestinal short-chain fatty acid level, the down-regulation of inflammation-associated cytokine secretion, a notable reduction in fecal CD toxin levels, and CD viable bacterial counts. Concurrently, there was an augmentation in the level of gut microbial diversity, accompanied by a rapid reduction in Enterococcus abundance. This ABB formulation holds promise for further development into a novel microecological formulation for the treatment of CDI.