The phloeodictine-based 6-hydroxy-2,3,4,6-tetrahydropyrrolo[1,2-<i>a</i>]pyrimidinium structural moiety with an <i>n</i>-tetradecyl side chain at C-6 has been demonstrated to be a new antifungal template. Thirty-four new synthetic analogues with modifications of the bicyclic tetrahydropyrrolopyrimidinium skeleton and the N-1 side chain have been prepared and evaluated for in vitro antifungal activities against the clinically important fungal pathogens including <i>Cryptococcus neoformans</i> ATCC 90113, <i>Candida albicans</i> ATCC 90028, <i>Candida glabrata</i> ATCC 90030, <i>Candida krusei</i> ATCC 6258, and <i>Aspergillus fumigatus</i> ATCC 90906. Nineteen compounds (<b>5</b>, <b>21</b>-<b>31</b>, <b>34</b>-<b>38</b>, <b>44</b>, and <b>48</b>) showed antifungal activities against the aforementioned five fungal pathogens with minimum inhibitory concentrations (MICs) in the range 0.88-10 μM, and all were fungicidal with minimum fungicidal concentrations (MFCs) similar to the respective MIC values. Compounds <b>24</b>, <b>36</b>, and <b>48</b> were especially active against <i>C. neoformans</i> ATCC 90113 with MIC/MFC values of 1.0/1.0, 1.6/1.6, and 1.3/2.0 μM but exhibited low cytotoxicity with an IC<sub>50</sub> > 40 μM against the mammalian Vero cells. The structure and antifungal activity relationship indicates that synthetic modifications of the phloeodictines can afford analogues with potent antifungal activity and reduced cytotoxicity, necessitating further preclinical studies of this new class of antifungal compounds.