The antimetabolite 5'-Fluorouracil (5FU) is an analog of uracil commonly employed as a chemotherapeutic agent in the treatment of a range of cancers including colorectal tumors. To assess the cellular effects of 5FU, we performed a genome-wide screening of the haploid deletion library of the eukaryotic model Schizosaccharomyces pombe. Our analysis validated previously characterized drug targets including RNA metabolism, but it also revealed unexpected mechanisms of action associated with chromosome segregation and organization (post-translational histone modification, histone exchange, heterochromatin). Further analysis showed that 5FU affects the heterochromatin structure (decreased levels of histone H3 lysine 9 methylation) and silencing (down-regulation of heterochromatic dg/dh transcripts). To our knowledge, this is the first time that defects in heterochromatin have been correlated with increased cytotoxicity to an anticancer drug. Moreover, the segregation of chromosomes, a process that requires an intact heterochromatin at centromeres, was impaired after drug exposure. These defects could be related to the induction of genes involved in chromatid cohesion and kinetochore assembly. Interestingly, we also observed that thiabendazole, a microtubule-destabilizing agent, synergistically enhanced the cytotoxic effects of 5FU. These findings point to new targets and drug combinations that could potentiate the effectiveness of 5FU-based treatments. 