The identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen, Aspergillus fumigatus, remains outstanding.Wefound that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway in A. fumigatus. Deletion of either pesL (pesL) or pes1 (pes1) resulted in complete loss of fumigaclavineCbiosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorginCand verruculogen, increased sensitivity toH2O2, and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion of pesL resulted in severely reduced virulence in an invertebrate infection model (P<0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster for A. fumigatus. Liquid chromatography/diode array detection/ mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in both A. fumigatus wild-type andpesL strains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesis in vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster crosstalk phenomenon, a theme which is beginning to emerge in the literature.