Effective cancer treatment remains challenging due to the genomic instability of tumors and the frequent emergence of resistance. Traditional approaches such as radiotherapy, chemotherapy, and immunotherapy face limitations in addressing tumor heterogeneity and resistance mechanisms. Targeting the DNA damage response (DDR) pathway has emerged as an innovative strategy, either as monotherapy or in combination with conventional treatments. DDR-targeted therapies, including poly-ADP-ribose polymerase (PARP) inhibitors, have shown promise in reducing tumor growth and enhancing patient outcomes. Emerging targets such as ATM, ATR, CHK1/2, WRN, and PARG, coupled with cutting-edge technologies like CRISPR and proteolysis-targeting chimeras (PROTACs), have opened new avenues for precise and effective cancer treatment. Furthermore, combining DDR inhibitors with established therapies, such as immune checkpoint inhibitors, has demonstrated synergistic benefits, improving therapeutic efficacy and overcoming resistance. Beyond cancer, DDR inhibitors also offer the potential to combat bacterial pathogens by exploiting vulnerabilities in microbial DNA repair systems. This review focuses on the major advantages, challenges, and future directions of DDR-targeted therapies in cancer and bacterial infections. We also discuss the integration of these therapies with traditional approaches, highlighting their potential to enhance therapeutic outcomes across diverse applications.