Systemic fungal infections pose a serious clinical problem. Treatment options are limited, and antifungal drug resistance is increasing. In addition, a substantial proportion of patients do not respond to therapy despite being infected with fungi that are susceptible to the drug. The discordance between overall treatment outcome and low levels of clinical resistance may be attributable to antifungal drug tolerance. In this Review, we define and distinguish resistance and tolerance and discuss the current understanding of the molecular, genetic and physiological mechanisms that contribute to those phenomena. Distinguishing tolerance from resistance might provide important insights into the reasons for treatment failure in some settings.