Cancer remains a leading cause of death worldwide, highlighting the urgent need for novel and more effective treatments. Natural products, with their structural diversity, represent a valuable source for the discovery of anticancer compounds. In this study, we screened 750 Antarctic extracts to identify potential inhibitors of human topoisomerase 1 (hTOP1), a key enzyme in DNA replication and repair, and a target of cancer therapies. Bioassay-guided fractionation led to the identification of palmitic acid (PA) as the active compound from the Antarctic sponge Artemisina plumosa, selectively inhibiting hTOP1. Our results demonstrate that PA irreversibly blocks hTOP1-mediated DNA relaxation and specifically inhibits the DNA religation step of the enzyme’s catalytic cycle. Unlike other fatty acids, PA exhibited unique specificity, which we confirmed through comparisons with linoleic acid. Molecular dynamics simulations and binding assays further suggest that PA interacts with hTOP1-DNA complexes, enhancing the inhibitory effect in the presence of camptothecin (CPT). These findings identify PA as a hTOP1 inhibitor with potential therapeutic implications, offering a distinct mechanism of action that could complement existing cancer therapies.