Bacteria and their metabolic products profoundly affect the immune system. Research has shown that early postnatal supplementation with specific probiotic strains, such as Escherichia coli O83:K24:H31 (EcO83), can provide health benefits. EcO83 is a facultative anaerobe capable of generating energy through multiple metabolic pathways, an adaptation that allows it to thrive in the gastrointestinal tract where oxygen availability can vary. Despite these advantages, the precise mechanisms through which this probiotic, and in particular its metabolites, functions remain largely unknown. In this study, we aimed to identify the metabolites that EcO83 produces when cultured under both aerobic and anaerobic conditions. We also aimed to investigate the immunomodulatory effects of these metabolites on human peripheral blood mononuclear cells (PBMCs), mouse splenocytes, and bone marrow-derived dendritic cells (BMDCs) in vitro. Our results revealed the presence of short-chain fatty acids (SCFA), namely acetate and propionate, in the supernatants of both aerobic and anaerobic EcO83 cultures. Interestingly, the supernatants from the aerobic cultures induced greater production of Th1 cytokines, such as IFN-γ, in PBMCs, whereas anaerobic culture supernatants more prominently triggered the Th2 cytokine IL-13. Similarly, murine splenocytes exhibited increased IFN-γ production when stimulated with aerobic supernatants. Additionally, both aerobic and anaerobic EcO83 supernatants promoted the release of proinflammatory (TNF-α) and anti-inflammatory (IL-10) cytokines from the BMDCs, as well as their maturation, evidenced by the upregulation of surface marker CD80. In conclusion, we are the first to report that EcO83 produces SCFA, including acetate and propionate, under varying oxygen conditions. Our findings suggest that probiotics can exert beneficial effects through their secreted metabolites, not solely through the presence of the bacterial cells themselves, making them a promising postbiotic solution for therapeutic administration.