In recent years the combined use of chemotherapy and immunotherapy, collectively termed chemoimmunotherapy, has emerged as a promising treatment option for patients with cancer. Antibiotics are commonly used to reduce infection-related complications in patients undergoing chemotherapy. Intriguingly, accumulating evidence has implicated gut microbiota as a critical determinant of host antitumor immune responses, raising the question as to whether the use of broad-spectrum antibiotics would invariably diminish tumor response to chemoimmunotherapies. We investigated the impact of antibiotics on the therapeutic outcomes of cyclophosphamide (CTX) chemotherapy and adoptive T-cell therapy (ACT) where CTX was used as the host-conditioning regimen in mice. We show that antibiotic prophylaxis dampened the endogenous T cell responses elicited by CTX, and reduced the efficacy of CTX against B-cell lymphoma. In the ACT setting, antibiotics administration impaired the therapeutic effects of adoptively transferred tumor-specific CD4+ T cells in mice with implanted colorectal tumors. In contrast, long-term antibiotic exposure did not affect the efficacy of ACT using CD19-targeting chimeric antigen receptor (CAR) T cells in mice with systemic B-cell lymphoma, although it correlated with prolonged CAR expression and sustained B-cell aplasia. Our study demonstrates that chemoimmunotherapies may have variable reliance on intestinal microbiota for T cell activation and function, and thus have different sensitivities to antibiotic prophylaxis. These findings may have implications for the judicial use of antibiotics in cancer patients receiving chemoimmunotherapies.