Background <p>The Arctic environment, characterized by extreme conditions, hosts a largely untapped reservoir of fungal communities that have adapted to these harsh conditions by producing specialized bioactive secondary metabolites. Among these, <i>Acremonium</i> species also remain underexplored, despite their potential to produce structurally diverse and biologically active secondary metabolites. This is largely due to difficulties in sampling in remote Arctic regions and limited research focus on fungi from such environments. This study aimed to use an integrated workflow combining metabolomics, chemometrics, and bioactivity screening to prioritize <i>Acremonium</i> strains for the identification of bioactive secondary metabolites. We applied this workflow to investigate six <i>Acremonium</i> strains associated with driftwood from the Arctic: <i>A. ellipsoideum</i> (F1, F2), <i>A. synnematoferum</i> (F3, F4, F5), and <i>A. multiramosum</i> (F6), aiming to identify cytotoxic secondary metabolites.</p> Results <p>The integrated metabolomics and chemometrics approach revealed unique chemical fingerprints for <i>A. ellipsoideum</i> (F1) and <i>A. synnematoferum</i> (F5) among the six strains. By further combining bioactivity screening results, strain F5 was prioritized for further detailed study. Five compounds were isolated from F5 and structurally elucidated as cyclic depsipeptides: destruxin-A4 chlorohydrin (<b>1</b>), trichomide D (<b>2</b>), destruxin-A5 (<b>3</b>), homodestruxin (<b>4</b>), and homodestcardin (<b>5</b>). All compounds exhibited cytotoxic effects against the tested cell lines but did not exhibit activity in the targeted bioassays against the kinase FLT3 or the phosphatase PTP1B. Cytotoxic destruxin-type compounds likely play a significant ecological role, as they have been reported to exhibit entomopathogenic, nematocidal, and phytotoxic effects.</p> Conclusion <p>The identification of five known cytotoxic destruxin-type depsipeptides from <i>A. synnematoferum</i> as a new source expands the chemical diversity known for this genus and underscores their potential for the development of cytotoxic agents. These findings highlight not only the value of Arctic fungi as a reservoir of bioactive compounds but also the necessity of deciphering the ecological roles of cytotoxic metabolites produced by these organisms as they adapt to extreme environments. Furthermore, this study highlights the effectiveness of multi-informative-driven strain prioritization in uncovering bioactive metabolites from new fungal sources, emphasizing the significance of exploring Arctic fungal diversity for its potential to enhance chemical diversity, contribute to drug development, and broaden our understanding of ecological roles.</p>