Background <p>Phenylspirodrimanes (PSDs) are a unique class of meroterpenoids that have been extensively studied due to their diverse biological activities. These metabolites are supposedly biosynthesized through the farnesylation of orsellinate. However, the molecular basis has not yet been elucidated.</p> Results <p>Herein, using a combination of genome mining, bioinformatic alignment, and gene deletion approach, we characterized the dedicated gene cluster <i>psd</i> responsible for biosynthesizing PSDs in the fungus <i>Stachybotrys</i> sp. CPCC 401591. RNA sequencing-based transcriptomic analyses broadened our understanding of the genetic basis, regulation, and mechanisms of PSDs biosynthesis. Furthermore, based on the chemical structures of PSD derivatives characterized and deduced gene functions of the cluster <i>psd</i>, a hypothetical metabolic pathway for biosynthesizing chartarlactam K (<b>1</b>) was proposed. These results revealed the underlying mechanism of phenylspirodrimanes generation, and these findings might facilitate downstream metabolic engineering studies of PSDs or the production of new chemical entities.</p> Conclusions <p>Genome discovery and gene deletion experiments unveiled a previously uncharacterized biosynthetic gene cluster <i>psd</i> involved in the biosynthesizing PSD derivatives in <i>Stachybotrys</i> sp. CPCC 401591. In addition, based on the gene cluster data and transcriptomic analyses, a hypothetical biosynthetic pathway of <b>1</b> was put forward.</p> Graphical Abstract <p></p>