Brr2 is an RNA helicase belonging to the Ski2-like subfamily and an essential component of spliceosome. Brr2 catalyzes an ATP-dependent unwinding of the U4/U6 RNA duplex, which is a critical step for spliceosomal activation. An HTS campaign using an RNA-dependent ATPase assay and initial SAR study identified two different Brr2 inhibitors, 3 and 12. Cocrystal structures revealed 3 binds to an unexpected allosteric site between the C-terminal and the N-terminal helicase cassettes, while 12 binds an RNA-binding site inside the N-terminal cassette. Selectivity profiling indicated the allosteric inhibitor 3 is more Brr2-selective than the RNA site binder 12. Chemical optimization of 3 using SBDD culminated in the discovery of the potent and selective Brr2 inhibitor 9 with helicase inhibitory activity. Our findings demonstrate an effective strategy to explore selective inhibitors for helicases, and 9 could be a promising starting point for exploring molecular probes to elucidate biological functions and the therapeutic relevance of Brr2.