Gain-of-function variants in the Na+-regulated K+ channel KCNT1 are associated to severe, drug-resistant forms of epilepsy. Quinidine has been used as a KCNT1 blocker, both in vitro and in vivo, though with conflicting clinical outcomes.To identify novel KCNT1 blockers, in the present work an in-house library of >800 compounds underwent a virtual screening based on docking energy scores and similarity to the quinidine bound conformation in a homology model of hKCNT1. Twenty molecules were selected and further screened in vitro using a Tl+-based fluorescent assay in KCNT1-expressing CHO cells; in this assay, Tl+ flux was activated by the KCNT1 opener loxapine and inhibited by quinidine. Five molecules (CPK4, 13, 16, 18 and 20) showed IC50s for KCNT1 blockade 20-35 times lower than quinidine. Patch-clamp electrophysiology experiments confirmed their higher potency for KCNT1 block and revealed that three of them (CPK16, 18 and 20) failed to affect hERG channel, whose block is linked to arrhythmogenic liability. In silico simulation and mutagenesis experiments identified F346 as a critical binding determinant, along with additional interactions. CPK20 showed the highest in vitro metabolic stability. Moreover, this compound was able to counteract gain-of-function effects prompted by 2 recurrent epilepsy-causing KCNT1 variants (G288S and A934T).Altogether, these data identified CPK20 as a novel, selective and potent KCNT1 blocker deserving further investigation in relevant models of KCNT1-associated epilepsy.