Species of Streptomyces are well known producers of diverse biological metabolites, and anthracyclines are one class of the typical antitumor antibiotics in drug studies. As one of the longest known antibiotics, anthracyclines have been received great attention frequently since its first discovery. Some of these anthracyclines have been used in medicine for the treatment of some cancers. Moreover, even in recent years, novel anthracyclines are constantly being discovered, such as aranciamycin anhydride. In our effort to search for new biological compounds from endophytes, a Streptomyces sp. YIM66403 producing special orange pigments caught our attention. Large-scale fermentation and subsequent fractionation of the extract of broth and mycelium proved that Streptomyces sp. YIM66403 was a strain yielding anthracyclines. As a result, we isolated a new anthracycline misamycin (1), together with η-pyrromycinone (2), 4-Deoxy-ε-pyrromycinone (3) and other six known anthracyclines including cinerubin A (4), cinerubin B (5), antibiotic 80334 C (6), cinerubin X (7), collinemycin (8) and musettamycin (9). Compound 1 showed cytotoxicity in the 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS) method and anti-Staphylococcus aureus activity in comparison with other antimicrobial activity. The structure of misamycin (1) was established by various spectroscopic methods, whereas the structures of other known compounds (2–9) were confirmed by comparing their 1H and 13C-NMR data with these of compounds in the literature. In this paper, we would like to report the isolation, structure determination and biological activity of 1.