Chimeric antigen receptor (CAR)-T cell therapy has transformed hematological cancer treatment, yet nearly half of patients still relapse or progress. Increasing evidence implicates the gut microbiome and antibiotic exposure as key modulators of clinical outcomes. In a cohort of 129 patients across three German centers, shotgun metagenomics and targeted mass spectrometry revealed that reduced short-chain fatty acids, particularly valeric acid, prior to CAR-T cell therapy correlated with increased risk of disease progression. Conversely, high levels of indole metabolites, including indole-3-carboxaldehyde and indole-3-acetic acid as well as the branched-chain fatty acid isovaleric acid, were linked to adverse outcomes. Functional validation in human and murine CAR-T cell models demonstrated that valeric acid supplementation enhanced, while indole-3-carboxaldehyde and isovaleric acid impaired, CAR-T cell efficacy. These findings reveal the opposing roles of immunomodulatory metabolites on CAR-T cell therapy, carrying significant implications for the design of metabolite-guided, microbiome-based therapeutics.