The etiological agent of the most dangerous form of malaria, Plasmodium falciparum, has developed resistance or reduced sensitivity to the majority of the drugs available to treat this deadly disease. Innovative antimalarial therapies are therefore urgently required. P. falciparum serine protease subtilisin-like protease 1 (PfSUB1) has been identified as a key enzyme for merozoite egress from red blood cells and invasion. We present herein the rational design, synthesis, and biological evaluation of novel and potent difluorostatone-based inhibitors. Our bioinformatic-driven studies resulted in the identification of compounds 1a, b as potent and selective PfSUB1 inhibitors. The enzyme/inhibitor interaction pattern herein proposed will pave the way to the future optimization of this class of promising enzyme inhibitors.