Heat shock protein 90 (HSP90) is an important molecular chaperone in plants. However, HSP90-mediated plant immune response remains elusive in cassava. In this study, cassava bacterial blight (CBB) induces the expression of MeHsf8, which directly targets MeHSP90.9 to activate its expression and immune response. Further identification of SHI-related sequence 1 (MeSRS1) and MeWRKY20 as MeHSP90.9 co-chaperones revealed the underlying mechanism of MeHSP90.9-mediated immune response. MeHSP90.9 interacts with MeSRS1 and MeWRKY20 to promote their transcriptional activation of salicylic acid (SA) biosynthetic gene avrPphB Susceptible 3 (MePBS3) and tryptophan metabolic gene N-acetylserotonin O-methyltransferase 2 (MeASMT2), respectively, so as to activate SA biosynthesis but inhibit tryptophan-derived auxin biosynthesis. Notably, genetic experiments confirmed that overexpressing MePBS3 and MeASMT2 could rescue the effects of silencing MeHsf8-MeHSP90.9 on disease resistance. This study highlights the dual regulation of SA and auxin biosynthesis by MeHSP90.9, providing the mechanistic understanding of MeHSP90.9 client partners in plant immunity.