ABSTRACT: The exopolysaccharide galactosaminogalactan (GAG) plays an important role in mediating adhesion, biofilm formation, and virulence in the pathogenic fungus Aspergillus fumigatus. Previous work showed that in A. fumigatus, the Lim domain-binding protein PtaB can form a complex with the sequence-specific transcription factor SomA for regulating GAG biosynthesis, biofilm formation, and asexual development. However, transcriptional coactivators required for biofilm formation in A. fumigatus remain uncharacterized. In this study, Spt20, an orthologue of the subunit of the Saccharomyces cerevisiae transcriptional coactivator Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, was identified as a regulator of biofilm formation and asexual development in A. fumigatus. The loss of spt20 caused severe defects in the GAG biosynthesis, biofilm formation, conidiation, and virulence of A. fumigatus. RNA sequence data demonstrated that Spt20 positively regulates the expression of the GAG biosynthesis genes uge3 and agd3, the developmental regulator medA, and genes involved in the conidiation pathway. Moreover, more than 10 subunits of the SAGA complex (known from yeast) could be immunoprecipitated with Spt20, suggesting that Spt20 acts as a structural subunit of the SAGA complex. Furthermore, distinct modules of SAGA regulate GAG biosynthesis, biofilm formation, and asexual development in A. fumigatus to various degrees. In summary, the novel biofilm regulator Spt20 is reported, which plays a crucial role in the regulation of fungal asexual development, GAG biosynthesis, and virulence in A. fumigatus. These findings expand knowledge on the regulatory circuits of the SAGA complex relevant for the biofilm formation and asexual development of A. fumigatus. IMPORTANCE Eukaryotic transcription is regulated by a large number of proteins, ranging from sequence-specific DNA-binding factors to transcriptional coactivators (chromatin regulators and the general transcription machinery) and their regulators. Previous research indicated that the sequence-specific complex SomA/PtaB regulates the biofilm formation and asexual development of Aspergillus fumigatus. However, transcriptional coactivators working with sequence-specific transcription factors to regulate A. fumigatus biofilm formation remain uncharacterized. In this study, Spt20, an orthologue of the subunit of the Saccharomyces cerevisiae Spt-Ada-Gcn5-acetyltransferase (SAGA) complex, was identified as a novel regulator of biofilm formation and asexual development in A. fumigatus. The loss of spt20 caused severe defects in galactosaminogalactan (GAG) production, conidiation, and virulence. Moreover, nearly all modules of the SAGA complex were required for the biofilm formation and asexual development of A. fumigatus. These results establish the SAGA complex as a transcriptional coactivator required for the biofilm formation and asexual development of A. fumigatus.