Gut-resident microbiota associate with host immune system to promote homeostasis, and regulatory T cells (Tregs) are critical in the maintenance of immune balance. Tregs have immunosuppressive activity, and their presence hampers the development of inflammatory diseases. This review aims to unravel microbiome impact on Tregs in bowel inflammation and harnessing such interaction to combat colitis as a separate disease or a consequence of immune checkpoint inhibitor (ICI) therapy of cancer. Short-chain fatty acids (SCFAs) are microbial-derived metabolites associated positively with Treg generation and maintenance and being effective for hampering bowel inflammation. Treg induction shapes gut microbiota profile and support microorganism colonization in their niche and protect the host from inflammation, while suppression of Treg differentiation and activity directs microbiota-induced Th17 expansion and inducing inflammation. Thus, balancing Treg representation with Th17 cells and Treg reprogramming through manipulation of gut microbiota can offer therapy. Microbiota epithelial attachment/detachment and interaction with antigen-presenting cells (APCs) are important for the final fate of T cell signature. Fecal microbial transplantation (FMT) is a strategy for promoting normobiosis and represents a navel approach to targeting colitis. FMT with appropriate microbiota from healthy donors can reinforce microbial diversity, density and persistence to enrich their environment with transforming growth factor (TGF)-β, induce IL-10 producing APCs and reinforce gut barrier, with all these being effective for recovering Tregs, restoring intestinal homeostasis and hampering colitis. ICI therapy of cancer may predispose subjects to colitis due to the impact on microbiome and reducing Treg population. FMT promotes local Treg reorchestration, being advantageous in cancer patients.