Rice blast is an essential factor affecting rice yield and quality, which is caused by Magnaporthe oryzae (M. oryzae). Isobavachalcone (IBC) is a botanical fungicide derived from the seed extract of the Leguminosae plant Psoralea corylifolia L. and has shown an excellent rice blast control effect in field applications. To explore the potential targets of rice blast control, the analysis of the differentially expressed proteins (DEPs) between the liquid culture medium of mycelium treated by 10 mg/L of IBC for 2 h and the control group indicated that Enolase 1 (ENO1) was the most significantly down-regulated DEP with a fold change value of 0.305. In vitro experiments showed that after treating liquid culture mycelium with 10 mg/L of IBC for 0.5, 1, 2, 4, and 8 h, the enzymatic activity of ENO1 in the IBC experimental groups was 0.97, 0.75, 0.52, 0.44, and 0.39 times as much as in the control groups, respectively. To further explore the molecular interaction and binding mode between IBC and ENO1, the three-dimensional structure of ENO1 was established based on homology modeling. Molecular docking and molecular dynamics simulation showed that IBC had a pi−pi stacking effect with the residue TYR_365, a hydrogen bond interaction with the residue ARG_393, and hydrophobic interactions with non-polar residues ALA_361, LYS_362, and VAL_371 of ENO1. These findings indicated that ENO1 is a potential target of M. oryzae, which would pave the way for screening novel effective fungicides targeting ENO1.