Methotrexate is a cytotoxic agent that is commonly used to treat autoimmune diseases, and both solid and hematological malignancies. Drug interactions are of particular concern during methotrexate therapy, as they have the potential to alter the pharmacokinetics of methotrexate and lead to significant toxicity. One agent that has the potential to interact with methotrexate is the antimicrobial agent trimethoprim with sulfamethoxazole. Consideration of this interaction is of importance in patients that are undergoing high‐dose methotrexate therapy for hematological malignancies as trimethoprim/sulfamethoxazole is the preferred agent for Pneumocystis jiroveci pneumonia prophylaxis. Current Australian guidelines recommend avoiding concurrent administration of high‐dose methotrexate and trimethoprim/sulfamethoxazole, yet evidence to support this recommendation is unclear. This review investigates the current available evidence that focuses on the interaction between high‐dose methotrexate and trimethoprim/sulfamethoxazole and aims provide guidance for the use of this combination in clinical practice.