A pair of new diastereomeric isoindolinone alkaloids, spirocollequins A and B, were isolated from an endophytic fungus, Colletotrichum boninense AM-12-2. Their structures are featuring a 4,5-dihydro-spiro[furan-2,1′-isoindolin]-3′-one core. Their structures were determined by 1D/2D NMR, the Mosher’s ester method, and DFT-GIAO NMR chemical shift calculations, coupled with advanced statistical procedures. Spirocollequins A and B exhibited in vitro antiplasmodial activity at the IC50 (μM) values of 9.72 ± 0.61 and 4.71 ± 0.77, respectively.