This study aimed to elucidate the complex interplay between microbiota diversity, microbial-derived signals, and alloimmune responses in the context of allograft rejection. At the whole-body microbiome level, a diverse microbial community fine-tuned the capacity of systemic antigen-presenting cells (APCs) to prime alloreactive T cells and orchestrate potent alloimmune responses. Targeting bacterial groups with broad-spectrum antibiotics diminished the microbiota diversity, dampened APC poising status, and weakened alloimmune responses, ultimately prolonging allograft survival. Furthermore, at the individual bacterium level, administration of Alistipes oderdonkii and Parabacteroides distasonis strains was sufficient to prolong allograft survival, suggesting their potential as probiotic candidates to enhance transplant outcomes. Conversely, colonization with a skin-restricted commensal, Staphylococcus epidermidis accelerated allograft rejection by augmenting the effector phase of the alloimmune response. This was achieved through enhancing skin APC activation of alloreactive T cells and triggering a host-versus-commensal T cell response. Lastly, at the microbial metabolite level, administration of butyrate inhibited quantitatively and qualitatively the myeloid cell compartment systemically, reducing the innate immune cell execution of the effector phase. These findings underscore the pivotal role of microbiota-derived signals and microbial modulation in alloimmune responses and graft outcomes.