The mycalamides belong to a family of protein synthesis inhibitors noted for antifungal, antitumour, antiviral, immunosuppressive, and nematocidal activities. Here we report a systematic analysis of the role of drug efflux pumps in mycalamide resistance and the first isolation of mycalamide E. In human cell lines, neither P‐glycoprotein overexpression nor the use of efflux pump inhibitors significantly modulated mycalamide A toxicity in the systems tested. In Saccharomyces cerevisiae, it appears that mycalamide A is subject to efflux by the principle mediator of xenobiotic efflux, Pdr5p along with the major facilitator superfamily pump Tpo1p. Mycalamide E showed a similar efflux profile. These results suggest that future drugs based on the mycalamides are likely to be valuable in situations where efflux pump–based resistance leads to failure of other chemotherapeutic approaches, although efflux may be a mediator of resistance in antifungal applications. © 2012 Wiley Periodicals, Inc. J Biochem Mol Toxicol 26:94–100, 2012; View this article online at wileyonlinelibrary.com. DOI 10.1002/jbt.20414