Human exonuclease 1 (EXO1), a member of the structure-specific nuclease family, plays a critical role in maintaining genome stability by processing DNA double-strand breaks (DSBs), nicks, and replication intermediates during DNA replication and repair. As its exonuclease activity is essential for homologous recombination (HR) and replication fork processing, EXO1 has emerged as a compelling therapeutic target, especially in cancers marked by heightened DNA damage and replication stress. Through high-throughput screening of 45,000 compounds, we identified seven distinct chemical scaffolds that demonstrated effective and selective inhibition of EXO1. Representative compounds from two of the most potent scaffolds, C200 and F684, underwent a comprehensive docking analysis and subsequent site-directed mutagenesis studies to evaluate their binding mechanisms. Biochemical assays further validated their potent and selective inhibition of the EXO1 nuclease activity. Tumor cell profiling experiments revealed that these inhibitors exploit synthetic lethality in BRCA1-deficient cells, emphasizing their specificity and therapeutic potential for targeting genetically HR-deficient (HRD) cancers driven by deleterious mutations in HR genes like BRCA1/2. Mechanistically, EXO1 inhibition suppressed DNA end resection, stimulated the accumulation of DNA double-strand breaks, and triggered S-phase PARylation, effectively disrupting DNA repair pathways that are essential for cancer cell survival. These findings establish EXO1 inhibitors as promising candidates for the treatment of HRD cancers and lay the groundwork for the further optimization and development of these compounds as targeted therapeutics.