Micromycetes from unexplored sources represent an opportunity to discover novel natural products to control insect pests. With this aim, a strain of Acremonium masseei CICY026 isolated from a tropical sinkhole was identified, cultured on fermented rice, and its ethyl acetate extract (EAE) was evaluated against three serious phytophagous insects (Bemisia tabaci, Myzus persicae, and Rhopalosiphum padi). DNA from A. masseei CICY026 was used to confirm its identity. EAE caused settling inhibition (SI) of M. persicae and R. padi (67.5% and 75.3%, respectively). Bioassay-guided fractionation of the active EAE led to the isolation of a novel metabolite, named hexahydroacremonintriol (1), and of acremonin A glucoside (2). The structures of 1 and 2 were determined using IR, one- and two-dimensional NMR, HRMS, and confirmed by theoretical data. The aphid M. persicae was noticeably sensitive to 1 and 2 (SI: 55.6% and 67.2%, respectively), whereas R. padi was only slightly affected by 1 (SI: 59%). This new knowledge about mycobiota from these special sinkhole ecosystems will inform the development of new biorational pesticides.