An original approach was undertaken to explore the metabolomic variability of macroscopic fungi collected in French Guiana. Extracts from 160 specimens, representing 13 fungal orders, were analyzed using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). The resulting metabolomic profiles enabled the construction of chemotaxonomic dendrograms, which successfully grouped most samples assigned to the order Xylariales and highlighted chemodiversity at both the genus and species levels within this lineage. Principal Component Analysis based on molecular family presence/absence revealed 35 discriminant clusters, including specific markers distinguishing Thamnomyces chordalis and Phylacia sp. Five previously undescribed natural products were isolated and structurally characterized: four polyketides-prochordapyrone (1) and chordapyrones A-C (2-4)-and one terpenoid, chordapyranone (5). Structural elucidation was accomplished through a combination of nuclear magnetic resonance (NMR) spectroscopy, circular dichroism and single-crystal X-ray diffraction. Based on structural and mechanistic considerations, a putative biosynthetic pathway was proposed for the formation of the polyketide compounds.