Heterochromatin is essential for regulating global gene transcription and protecting genome stability, and may play a role in tumor suppression. Drugs promoting heterochromatin are potential cancer therapeutics but very few are known. In order to identify drugs that can promote heterochromatin, we used a cell-based method and screened NCI drug libraries consisting of oncology drugs and natural compounds. Since heterochromatin is originally defined as intensely stained chromatin in the nucleus, we estimated heterochromatin contents of cells treated with different drugs by quantifying the fluorescence intensity of nuclei stained with Hoechst DNA dye. We used HeLa cells and screened 231 FDA-approved oncology and natural substance drugs included in two NCI drug libraries representing a variety of chemical structures. Among these drugs, streptonigrin most prominently caused an increase in Hoechst-stained nuclear fluorescence intensity. We further show that streptonigrin treated cells exhibit compacted DNA foci in the nucleus that co-localize with Heterochromatin Protein 1 alpha (HP1α), and exhibit an increase in total levels of the heterochromatin mark, H3K9me3. Interestingly, we found that streptonigrin promotes heterochromatin at a concentration as low as one nanomolar, and at this concentration there were no detectable effects on cell proliferation or viability. Finally, in line with a previous report, we found that streptonigrin inhibits STAT3 phosphorylation, raising the possibility that non-canonical STAT function may contribute to the effects of streptonigrin on heterochromatin. These results suggest that, at low concentrations, streptonigrin may primarily enhance heterochromatin formation with little toxic effects on cells, and therefore might be a good candidate for epigenetic cancer therapy.