There are no targeted therapies available for triple-negative breast cancers (TNBCs) in part because they represent a heterogeneous group of tumors with diverse oncogenic drivers. Our goal is to identify targeted therapies for subtypes of these cancers using a mechanism-blind screen of natural product extract libraries. An extract from Desmanthodium guatemalense was 4-fold more potent for cytotoxicity against MDA-MB-231 cells, which represent the mesenchymal stem-like (MSL) subtype, as compared to cells of other TNBC subtypes. Bioassay-guided fractionation led to the isolation of six polyacetylenes, and subsequent investigations of plant sources known to produce polyacetylenes yielded six additional structurally related compounds. A subset of these compounds retained selective cytotoxic effects in MSL subtype cells. Studies suggest that these selective effects do not appear to be due to PPARγ agonist activities that have previously been reported for polyacetylenes. A CRISPR-Cas9-mediated gene knockout screen was employed to identify the mechanism of selective cytotoxic activity of the most potent and selective compound, dehydrofalcarinol (1a). This genomic screen identified HSD17B11, the gene encoding the enzyme 17β-hydroxysteroid dehydrogenase type 11, as a mediator of the selective cytotoxic effects of 1a in MDA-MB-231 cells that express high levels of this protein. The Project Achilles cancer dependency database further identified a subset of Ewing sarcoma cell lines as highly dependent on HSD17B11 expression, and it was found these were also highly sensitive to 1a. This report demonstrates the value of CRISPR-Cas9 genome-wide screens to identify the mechanisms underlying the selective activities of natural products.