A pair of novel enantiomeric polyketide dimers, (+)- and (-)-ascomlactone A (1a and 1b), were obtained from a mangrove endophytic fungus Ascomycota sp. SK2YWS-L. The structures were elucidated based on spectroscopic methods, and the absolute configurations were determined by X-ray diffraction and electronic circular dichroism (ECD) calculations. Ascomlactone A possessed an unprecedented polymerization system, which constructed an unusual nine-membered lactone ring between the monomers. A possible biogenetic pathway was proposed. Both 1a and 1b exhibited significant inhibitory effects against α-glucosidase with IC50 values of 63.7 and 27.9 μM, respectively. A further docking study provided an inside perspective of the action in α-glucosidase.