Fructose-1,6-bisphosphate aldolase (FBA) represents an attractive new antifungal target. Here, we employed a structure-based optimization strategy to discover a novel covalent binding site (C292 site) and the first-in-class covalent allosteric inhibitors of FBA from <i>Candida albicans</i> (CaFBA). Site-directed mutagenesis, liquid chromatography-mass spectrometry, and the crystallographic structures of APO-CaFBA, CaFBA-<b>G3P</b>, and C157S-<b>2a4</b> revealed that S268 is an essential pharmacophore for the catalytic activity of CaFBA, and L288 is an allosteric regulation switch for CaFBA. Furthermore, most of the CaFBA covalent inhibitors exhibited good inhibitory activity against azole-resistant <i>C. albicans</i>, and compound <b>2a11</b> can inhibit the growth of azole-resistant strains 103 with the MIC<sub>80</sub> of 1 μg/mL. Collectively, this work identifies a new covalent allosteric site of CaFBA and discovers the first generation of covalent inhibitors for fungal FBA with potent inhibitory activity against resistant fungi, establishing a structural foundation and providing a promising strategy for the design of potent antifungal drugs.