Rho GTPases Cdc42 and RacA exhibit significant sequence homology and are conserved across eukaryotic species. These proteins function as molecular switches within various signal transduction pathways by cycling between GTP-bound (active) and GDP-bound (inactive) states. However, their specific functions in Aspergillus flavus remain largely unexplored. In this study, CRISPR/Cas9 system utilizing 5S rRNA and tRNA-gRNA tandem arrays was developed, achieving over 95% single-gene and 75% double-gene editing efficiencies. Phenotypic and transcriptome analyses revealed significant functional redundancy between cdc42 and racA. In the mutants, conidia germination was markedly accelerated, with early germination driven by increased hydrolase activity and ATP levels. The loss of either cdc42 or racA resulted in reduced pathogenicity, compromised cell wall integrity, diminished aflatoxin production, and disrupted oxidative systems. These proteins regulate the generation of ROS through their interaction with NoxR, the regulatory subunit of NADPH oxidase (Nox). Cdc42 and RacA exhibited opposing roles in fatty acid β-oxidation and pyruvate metabolism. The simultaneous loss of function in both genes is lethal, as evidenced by the inability of the ∆cdc42racA_tetOn mutant to grow on plates lacking doxycycline. The study reveals the critical roles of the closely related genes cdc42 and racA in the growth, development, and metabolism of A. flavus. These findings identify potential targets for mitigating the harmful effects of A. flavus and aflatoxins.