The present clinical scenario is one of the growing numbers of immunocompromised patients infected with a variety of fungal pathogens. AIDS, tuberculosis, immunosuppressive therapy, cancer chemotherapy, or the use of broad-spectrum antibiotics contribute to the boost of such patient category. However, progress in the treatment of fungal infections has been slow. Different polyenes and/or azoles are available for the treatment of systemic fungal infections, but it is becoming clear that new and more effective antifungal agents are urgently required. Fortunately, biosynthetic gene clusters for several antifungal polyene macrolides have been characterized, and this opened the way to generate improved antifungal compounds via genetic engineering as well as to understand the molecular mechanisms that regulate polyene production. Here, we will review the history of the discovery of antifungal polyenes, their environmental role, and the strategies currently used for their identification and improvement.