Parthenolide, a natural product from the feverfew plant and member of the large family of sesquiterpene lactones, exerts multiple biological and therapeutic activities including anti-inflammatory and anti-cancer effects. Here, we further study the parthenolide mechanism of action using activity-based protein profiling-based chemoproteomic platforms to map additional covalent targets engaged by parthenolide in human breast cancer cells. We find that parthenolide, as well as other related exocyclic methylene lactone-containing sesquiterpenes, covalently modify cysteine 427 of focal adhesion kinase 1 (FAK1), leading to impairment of FAK1-dependent signaling pathways and breast cancer cell proliferation, survival, and motility. These studies reveal a functional target exploited by members of a large family of anti-cancer natural products.