Abstract: Although anisotropic NMR spectroscopy has emerged as a powerful method for determining the relative configuration of complex natural products, major challenges persist with structurally flexible molecules. In this study, we conducted a systematic comparative analysis of stereochemical elucidation, combining anisotropic NMR spectroscopy and density functional theory (DFT) calculations on spiroepicoccin B (1) and epicoccin V (2), which were characterized as thiodiketopiperazine marine natural products isolated from the deep-sea-derived fungus Epicoccum nigrum SD-388. For the flexible compound 2, we compared various conformational sampling approaches, including an assessment of the quality of relative energies within the obtained ensembles. We demonstrated the critical role of dispersion correction within DFT computations to precisely account for weak non-bonded intramolecular interactions. By integrating anisotropic NMR analysis, chemical shifts, electronic circular dichroism, and DFT computations, we determined the absolute configurations and conformational ensembles for 1 and 2, respectively, highlighting the significance of the intramolecular methyl–π interaction in stabilizing one of the conformers. Our study introduces new strategies to address conformational flexibility in the stereochemical elucidation of challenging organic molecules.