Significance Cryptococcus neoformans is an opportunistic human pathogen, causing cryptococcal meningitis in immunocompromised individuals, with a mortality rate of more than 50%. We previously found that the Prp8 intein was a viable drug target for C. neoformans . In an effort to find new therapies, we developed screening assays and identified a small molecule and its fluoride derivative as potent Prp8 intein-splicing inhibitors. These inhibitors bind covalently to an intein active-site residue and inhibit protein splicing, leading to nonfunctional Prp8, which is essential for fungal growth. These molecules inhibited growth of C. neoformans and showed synergistic or additive effects with Food and Drug Administration-approved antimycotics. Overall, the identification of these intein-splicing inhibitors opens up prospects for a new class of antifungals.