The soil-borne, rhizosphere-competent, filamentous fungus Trichoderma virens is a well-known biocontrol agent able to control pathogenic fungi through the production of antibiotics, the induction of systemic resistance in host plants, or by directly parasitizing the competing fungus. Competition for iron is another means by which Trichoderma can hinder competing microorganisms, and siderophores are a means by which microorganisms obtain iron. In silico analysis of the T. virens genome suggested that two genes putatively encoding extracellular siderophore-producing nonribosomal peptide synthetases (NRPSs) were present. In this study, a disruption was created in one of the genes, TvNPS6, to create a mutant unable to produce the NRPS TvNps6 (DeltaTvnps6). Previously, a mutant (DeltaTvsidD) had been generated with a disruption in the second gene (TvSIDD) encoding an NRPS thought to be involved in siderophore biosynthesis. A double mutant (DeltaDeltaTvsidDTvnps6) was generated by transformation of a DeltaTvsidD strain with a vector targeting disruption of TvNPS6. This resulted in transformants disrupted within both the putative siderophore-producing NRPSs. Thus, three mutants were available for analysis of the role of these genes in the ecology of T. virens. Transformants were confirmed by PCR and Southern blotting analysis. Phenotypic characterization of the mutants included both HPLC analysis of siderophore production, growth on agar and in liquid media, conidiation, germination in the presence of hydrogen peroxide, biocontrol against Pythium ultimum, in vitro confrontation against Rhizoctonia solani and growth with iron chelators to determine the contribution of reductive iron assimilation (RIA) compared to that of siderophores. The HPLC analysis demonstrated that T. virens Gv 29-8 (wild-type) produced a single siderophore peak when grown in an iron-depleted medium. This peak was not present in the DeltaTvnps6 and DeltaDeltaTvsidDTvnps6 mutants but was apparent with the DeltaTvsidD mutants. From the HPLC analysis, T. virens evidently produces a coprogen-type siderophore. Few differences were observed in the other phenotypic tests, though hydrogen peroxide showed some small inhibitory effects towards the DeltaTvnps6 mutants. The addition of chelators, which inhibit RIA, exerted some negative effects on all strains growing under iron-limited media, particularly the DeltaTvnps6 and DeltaDeltaTvsidDTvnps6 strains. This study demonstrated that although T. virens has two genes putatively encoding siderophore producing NRPSs, only the TvNPS6 gene was required for extracellular siderophore production. The greater sensitivity of the mutants towards the iron chelators suggests that unlike other other fungi studied, Trichoderma virens utilizes RIA, rather than siderophore production, as the primary means by which the fungus obtains iron in an iron-limited environment.