A new hypothetic biosynthesis of the tricyclic spiroketal core of ascospiroketals A and B is proposed, which guided the development of a novel synthetic strategy for the asymmetric total synthesis of ent-ascospiroketals A and B. The synthesis features an efficient ring contraction rearrangement of the 10-membered lactone to the tricyclic spiroketal cis-fused γ-lactone core, which served as the common intermediate for the synthesis of both ent-ascospiroketals A and B through the Stille coupling reaction at the final step. In addition, seven diastereomers were prepared to conclusively confirm the structure of ent-ascospiroketal B.