The availability of new antifungal agents with unique mechanisms of action and improved tolerability has widened the possibilities for the use of combination antifungal therapy for difficult-to-treat opportunistic mycoses. However, the use of this therapy is largely governed by empiricism, especially in patients with invasive mould infections, for whom there is a tremendous need to improve outcomes. Because of the difficulties associated with the design and conduct of clinical trials of combination antifungal therapy for opportunistic mycoses, the majority of the studies evaluating antifungal combinations are still performed in the laboratory or using animal models of infection. However, the methods used to assess combined antifungal effects in vitro and in animals are poorly standardized, and there is little evidence that data generated from these studies can be translated in treating human mycotic infections. Despite the empiricism of combination antifungal therapy, certain principles help guide the use and study of these regimens.