Abstract Naphthoquinones are naturally occurring metabolites with recognized anti-cancer potential but limited clinical application. This study investigated the molecular mechanism of 8-hydroxy-2-(2-thenoyl)naphtho[2,3- b ]thiophene-4,9-dione ( 1 ), a new candidate for colorectal cancer (CRC) treatment, using different experimental settings: MTT, clonogenic, wound healing, and cell cycle assays; as well as RNA sequencing. Naphthoquinone 1 selectively reduced the viability and migration of HT-29 cells by G2/M arrest and changes in their transcriptome signature with significant effect on cellular survival, proliferation, angiogenesis, response to interferon, oxidative stress, and immune response. Impact analysis identified ferroptosis and MAPK pathways as significantly affected. In summary, our results suggest that 1 induces the selective death of CRC cells by inducing oxidative stress, ferroptosis, and MAPK inhibition.