Fungal endophytes are sources of novel bioactive compounds but relatively few agriculturally important fruiting plants harboring endophytes have been carefully studied. Previously, we identified a griseofulvin-producing
Xylariaspecies isolated from Vaccinium angustifolium, V. corymbosum, and Pinus strobus. Morphological and genomic analysis determined that it was a new species, described here as Xylaria ellisii. Untargeted high-resolution LC-MS metabolomic analysis of the extracted filtrates and mycelium from 15 blueberry isolates of this endophyte revealed differences in their metabolite profiles. Toxicity screening of the extracts showed that bioactivity was not linked to production of griseofulvin, indicating this species was making additional bioactive compounds. Multivariate statistical analysis of LC-MS data was used to identify key outlier features in the spectra. This allowed potentially new compounds to be targeted for isolation and characterization. This approach resulted in the discovery of eight new proline-containing cyclic nonribosomal peptides, which we have given the trivial names ellisiiamides A-H. Three of these peptides were purified and their structures elucidated by one and two-dimensional nuclear magnetic resonance spectroscopy (1D and 2D NMR) and high-resolution tandem mass spectrometry (HRMS/MS) analysis. The remaining five new compounds were identified and annotated by high-resolution mass spectrometry. Ellisiiamide A demonstrated Gram-negative activity against Escherichia coliBW25113, which is the first reported for this scaffold. Additionally, several known natural products including griseofulvin, dechlorogriseofulvin, epoxy/cytochalasin D, zygosporin E, hirsutatin A, cyclic pentapeptides #1–2 and xylariotide A were also characterized from this species.