Filamentous fungi have a long-standing tradition as industrial producers of primary and secondary metabolites. Initially, industrial scientists selected production strains from natural isolates that fulfilled both microbiological and technical requirements for economical production processes. Subsequently, genetically modified strains with novel properties were obtained through traditional strain improvement programs relying mostly on random mutagenesis. In recent years, however, recombinant technologies have contributed significantly to improve the capacities of production and have also allowed the design of genetically manipulated strains. These major advances were only made possible by basic research bringing deeper and novel insights into cellular and molecular fungal processes, thus allowing the design of genetically manipulated strains. This better understanding of fundamental genetic processes in model organisms has resulted in the design and generation of new experimental transformation strategies to manipulate specifically gene expression and function in diverse filamentous fungi, including those having a biotechnical significance. In this review, we summarize recent developments in the application of homologous DNA recombination and RNA interference to manipulate fungal recipients for further improvement of physiology and development in regards to biotechnical and pharmaceutical applications.