Multidrug transporters are membrane proteins that are able to expel a broad range of toxic molecules from the cell. In humans, the overexpression of the multidrug resistance P-glycoprotein (Pgp) and the multidrug resistance-associated protein MRP1 (MRP) is a principal cause of resistance of cancers to chemotherapy. These multidrug transporters belong to the ATP-binding cassette (ABC) family of transport proteins that utilize the energy of ATP hydrolysis for activity. In microorganisms, multidrug transporters play an important role in conferring antibiotic resistance on pathogens. In the last decade, homologs of human Pgp and MRP have been found in microorganisms such as Plasmodium falciparum, Candida albicans, Saccharomyces cerevisiae and, more recently, in Lactococcus lactis. In this review, we will summarize the current state of knowledge on three major aspects of microbial ABC-type multidrug transporters: (i) the functional and structural similarities among these proteins in prokaryotic and eukaryotic cells, (ii) the molecular mechanism of these transporters, and (iii) their potential physiological role.