The genus Xylaria is a promising source of bioactive compounds. This study explored the diversity of secondary metabolites (SMs) in Xylaria longipes under three fermentation conditions, isolating 14 new SMs and 27 known ones. The structures were elucidated using NMR, HRESIMS, ROESY, and J-based configuration analysis, with absolute configurations determined through calculated ECD and ¹³C NMR data. A comprehensive library of SMs was constructed, facilitating metabolomics-wide association studies that identified culture conditions as a critical factor influencing SM production. This compound library also simplifies the determination of absolute configurations for diplosporins by analyzing J values and CD trends. Anti-proliferative assays against induced T/B lymphocytes and HaCaT cells revealed that more than half of the compounds exhibited significant inhibitory activity, with compounds 2, 15, and 32 reducing IFN-γ secretion. Compound 32 showed promising anti-psoriatic effects by inhibiting NF-κB p65 phosphorylation in HaCaT cells. This first systematic chemical study of X. longipes under varied conditions provides insights into structure-activity relationships.