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Colorectal cancer is ranked the third most cases and second cause of death in the world 2018. Many innovative anticancer drugs have been developed through a variety of natural bioactive compounds, one of which is from microorganisms. This study aims to prove the effect of ethyl acetate extract fraction of Streptomyces hygroscopicus Subsp. Hygroscopicus. in decreasing the expression of DLD-colorectal cancer DNA. The fractionation process used Flash Column Chromatography (BUCHI Reveleris PREP Purification System) while the percentage of nuclear DNA expression was measured using immunofluorescent (DAPI). Thirty fractions were obtained from the fractionation process. The DLD-1 colorectal cancer cell line was cultured and exposed to each fractions at doses of 80, 8, and 0.8 ppm for 2x24 hours. From immunofluorescent analysis, fractions 13, 15, 16, 18, and 19 are able to reduce the DAPI expression of DLD-1 colorectal cancer cell line below 100%. Statistical analysis shows differences in the percentage of nuclear DNA expression between the five fractions and controls (p = 0,000). The results of the Post Hoc Tukey HSD test shows that there are differences in the percentage of nucleus DNA expression between various dosages of the five fractions and controls (p <0.005). Correlation test shows that there is a correlation (p <0.05) between increasing the dose of the five fractions with the expression of nucleus DNA with a very strong correlation coefficient. So, it can be concluded that the administration of the five ethyl acetate extracts of S. hygroscopicus can reduce the proliferation ability as measured by the percentage of DNA expression of the DLD-1 colorectal cancer cell line nucleus cancer.