Four new bislactones, dihydroacremonol (1), clonostachyone (2), acremodiol B (3), and acremodiol C (4), along with one known compound, hymeglusin (5), were isolated from cultures of two fungal strains (MSX59876 and MSX59260). Both strains were identified based on phylogenetic analysis of molecular data as Clonostachys spp.; yet, they biosynthesized a suite of related, but different, secondary metabolites. Given the challenges associated with elucidating the structures and configurations of bislactones, GIAO NMR calculations were tested as a complement to traditional NMR and HRESIMS experiments. Fortuitously, the enantiomer of the new natural product (4) was known as a synthetic compound, and the predicted configuration from GIAO NMR calculations (i.e., for the relative configuration) and optical rotation calculations (i.e., for the absolute configuration) matched those of the synthesis product. These results engendered confidence in using similar procedures, particularly the mixture of GIAO NMR shift calculations coupled with an orthogonal technique, to predict the configuration of 1-3; however, there were important limitations, which are discussed for each of these. The metabolites displayed antimicrobial activities, with compounds 1 and 4 being the most potent against Staphylococcus aureus with MICs of 1 and 4 μg/mL, respectively.