Glioblastoma (GBM) is the most common and lethal brain tumor. GBM has a remarkable degree of motility and is able to infiltrate the healthy brain. In order to perform a rationale-based drug-repositioning study, we have used known inhibitors of two small Rho GTPases, Rac1 and Cdc42, which are upregulated in GBM and are involved in the signaling processes underlying the orchestration of the cytoskeleton and cellular motility. The selected inhibitors (R-ketorolac and ML141 for Cdc42 and R-ketorolac and EHT 1864 for Rac1) have been successfully employed to reduce the infiltration propensity of GBM in live cell imaging studies. Complementarily, all-atom simulations have elucidated the molecular basis of their inhibition mechanism, identifying the binding sites targeted by the inhibitors and dissecting their impact on the small Rho GTPases' function. Our results demonstrate the potential of targeting the Rac1 and Cdc42 proteins with small molecules to contrast GBM infiltration growth and supply precious information for future drug discovery studies aiming to fight GBM and other infiltrative cancer types.