Mesenchymal stem cells (MSCs) are multipotent stromal cells residing in various tissue niches, widely recognized for their regenerative and immunomodulatory properties. They play a pivotal role in promoting wound healing, regulating inflammatory immune responses, and exerting antibacterial activity against a broad range of pathogens. These antimicrobial effects are mediated either directly, via the secretion of bioactive molecules and antimicrobial peptides (AMPs), or indirectly, by modulation of immune and inflammatory pathways. Recent evidence underscores the significance of the bidirectional interaction between MSCs and microorganisms. This crosstalk not only shapes the antimicrobial function of MSCs but also influences critical aspects of MSCs’ biology, including their proliferation, differentiation, immunological behavior, and tissue-regenerative capacity. However, despite growing interest, the underlying mechanisms of microbe–MSC interactions remain poorly understood, representing a major gap in our current knowledge. This chapter aims to bridge that gap by exploring the complex dialogue between MSCs and the microbiota, emphasizing how microbial signals modulate MSC fate and function, sometimes to the benefit of microbial survival and persistence. We also examine how disruptions in host–microbe homeostasis (dysbiosis) may impair MSC-mediated repair mechanisms and contribute to chronic disease progression. Notably, we highlight emerging evidence showing that microbiota-derived metabolites, such as polyamines, short-chain fatty acids (SCFAs), and indole derivatives, can significantly influence the therapeutic potential of MSCs following transplantation. Finally, we explore current and emerging clinical applications of MSCs and their secretome, including AMPs, exosomes, cytokines, and growth factors, in the fight against antimicrobial-resistant bacterial infections. By shedding light on the role of microbial mediators in MSC-based therapies, this chapter offers new insights into optimizing cell-based interventions in the face of escalating antimicrobial resistance.