Fungal infections continue to rise because of environmental pollution, increased radiation background, irrational use of broad-spectrum antibiotics, extensive use of cytostatics and immunosuppressants, and several other factors. Invasive mycoses are becoming more problematical for medical practice because of the rising population of immunocompromised patients. Polyene macrolide antibiotics (PMA) such as amphotericin B, nystatin, pimaricin, levorin, candicidin, and others are widely used in medical mycology for treatment of both surface and deep mycoses. However, PMA used in antifungal therapy do not fully meet the needs of physicians because of their limited efficacy due to their low water solubility, high toxicity, and emergence of resistant species of pathogenic fungi. Hence, the search for novel PMA derivatives with improved medical and biological properties is a very urgent target. In this report, we summarized our investigations on synthesis of various semi-synthetic PMA derivatives that were prepared by chemical modification. It was found that chemical modification, in particular phosphorylation, allows preparing highly efficient PMA derivatives with low toxicity and extended spectrum of biological activity. Biological studies have shown that synthesized semi-synthetic PMA derivatives possessed high antifungal activity against different species of pathogenic fungi and were 3-6 times less toxic than the starting antibiotics. The additional virological tests have demonstrated that semi-synthetic derivatives of levorin, nystatin, amphotericin B, mycoheptin, and lucensomycin exhibited high antiviral activity against DNA-containing vaccinia virus and RNA-containing oncogenic Rous sarcoma virus and type A and B influenza viruses. The results obtained for hydrophosphoryl derivatives of PMA, concerning their antiviral action against the RNA-containing Rous sarcoma virus, were especially interesting because these experimental data were proposed as a retrovirus model suitable for screening and studying of anti-AIDS drugs. It was established that the obtained semi-synthetic PMA derivatives were effective against many resistant strains of pathogenic fungi. Thus, dialkyl(diaryl)amidophosphate derivatives of amphotericin B and 3-N-a-dialkoxy(diphenoxy)phosphonate derivatives of pimaricin indicated high antifungal activity against several resistant strains of pathogenic fungi such as Candida albicans, Aspergillus fumigatus, and Cryptococcus neoformans, which cause opportunistic fungal infections occurring in AIDS patients. In present report, a special attention is paid to an evaluation of structure-activity relationships for synthesized semi-synthetic PMA derivatives. Possible mechanisms of action of these new powerful antimycotics on pathogenic fungi are considered. The prospects of application in medical practice of most effective PMA derivatives (lead compounds), prepared by us, will be discussed.