Chromatin regulation is commonly disrupted in human cancers. The identification of chemical probes that reverse chromatin defects can provide mechanistic insights as well as therapeutic opportunities for these cancers. One such cancer is Ewing sarcoma, a pediatric bone tumor characterized by the EWSR1-FLI1 fusion protein. EWSR1-FLI1 activates an oncogenic transcriptional program by remodeling chromatin at characteristic genomic loci. To identify inhibitors of oncoprotein-mediated chromatin modulation, we adapted formaldehyde-assisted isolation of regulatory elements (FAIRE) for high-throughput use and screened a library of small molecules designed to inhibit a wide range of chromatin regulators. Among the compounds that selectively decreased chromatin accessibility at EWSR1-FLI1 binding sites, the class of histone deacetylase inhibitors was highly enriched. We found that treatment with these HDAC inhibitors altered both EWSR1-FLI1 transcript and protein levels. In addition, we identified a novel small molecule inhibitor of chromatin accessibility, UNC0621. This compound interacts directly with chromatin through nuclear protein intermediates. UNC0621 also inhibits the proliferation of Ewing sarcoma cells by inducing a cell cycle arrest. These data demonstrate the effect of chromatin-targeted small molecules on the biology of Ewing sarcoma cells, and validate the potential of chromatin-based assays to screen for compounds with therapeutic potential in cancers with epigenetic alterations.