Fungi belonging to the genus of Trichoderma have a long history of infecting crops of edible fungi and reducing the yield. Trichoderma aggressivum is the main causal agent of green mold disease in Agaricus bisporus. Despite its infamous role as a persistent and aggressive contamination in A. bisporus crops, T. aggressivum can also be used as a biocontrol agent or as a promoter of plant growth. In order to work efficiently with T. aggressivum on a molecular biology level, a transformation protocol is required. This study provides a detailed protocol on how to perform a transformation in T. aggressivum using plasmid DNA for ectopic integration. In addition, a Cas9-RNP-based approach has been established for genome editing. We performed two transformations to confirm the usability of the hph gene as well as the pyr4 gene from Trichoderma reesei as selection markers. First, we integrated the hph gene ectopically and determined the overall transformation efficiency. In the second transformation experiment, we disrupted the ornithine-5' phosphate decarboxylase gene of T. aggressivum by using the CRISPR-Cas9 approach. In total, four candidates showed the expected uridine auxotrophy and resistance to 5-FOA. Additionally, the genomic locus around the CRISPR target sites was sequenced to determine the exact circumstances around the gene disruption. To complete the genetic toolbox for T. aggressivum, the pyr4 gene of T. reesei was tested as a suitable selection marker in one of the generated uridine auxotrophic strains.IMPORTANCEResearchers need an efficient tool for genomic manipulation to investigate the fundamental biology of mycoparasitism of T. aggressivum and its correlation to secondary metabolites. We provide a protocol for transformation of T. aggressivum and successfully demonstrated transformation of T. aggressivum using a plasmid and genome editing applying a Cas9-RNP-based strategy. Simultaneously, we established two selection markers, the hph gene and pyr4 gene from T. reesei. By applying these methods, we give researchers the tools needed to investigate T. aggressivum on a deeper level. Possible applications include activation of biosynthetic gene clusters of secondary metabolites to determine the biosynthetic pathway and biotechnological applications of these compounds.