A reduction in the strong immunosuppressive activity of FK506 (<b>1</b>) is essential for developing this compound as an antifungal agent. Seven new FK506 analogues modified at both the FK506-binding protein 12- and the calcineurin-binding regions were biosynthesized. 9-DeoxoFK520 (<b>7</b>) exhibited a >900-fold reduction in the <i>in vitro</i> immunosuppressive activity but maintained significant antifungal activity, indicating that the C-9 and C-21 positions are critical for separation of immunosuppressive and antifungal activities. <b>7</b> exhibited robust synergistic antifungal activity with fluconazole. FK506 (<b>1</b>) is a 23-membered macrolide produced by several <i>Streptomyces</i> species and is used as an immunosuppressive drug to prevent the rejection of transplanted organs. FK506 has also exhibited antifungal, neuroprotective, and neuroregenerative activities. In humans, FK506 binds to FK506-binding protein (FKBP) 12, and the resulting FKBP12-FK506 complex interacts with a Ca<sup>2+</sup>-calmodulin-dependent phosphatase, calcineurin (CaN). Inactivation of CaN by forming the FKBP12-FK506-CaN ternary complex prevents the activation of nuclear factor of activated T cells (NF-AT), inhibiting the production of interleukin-2 and subsequent T-cell proliferation. This CaN signaling pathway also plays a critical role in the growth and pathogenesis of major fungal pathogens such as <i>Cryptococcus neoformans</i>, <i>Candida albicans</i>, and <i>Aspergillus fumigatus</i>. Therefore, the synthesis of FK506 analogues that can discriminate human FKBP12/CaN from its fungal counterparts may separate antifungal activity from the immunosuppressive activity, thereby allowing the development of a novel antifungal agent.