We have established a convenient and practical bioassay system for the screening of novel antiphage compounds and previously found pyrrolobenzodiazepine antibiotics RK-1441A and B, as well as depsipeptide antibiotics enopeptin A and B. In continuation of our research, we isolated a new compound RK-1009 (1) and two known compounds, factumycin (2) and its stereoisomer at the 14,15-double bond A73A (3), from an actinomycete identified as Streptomyces sp. isolated from a soil sample collected in Tsuruoka City, Yamagata Prefecture, Japan. The structure of RK-1009 was confirmed as a methoxy derivative of 3 at the position 28 based on spectral data including NMR, UV, IR, and MS. RK-1009 (1) inhibited the plaque formation of bacteriophage B at the concentration of 0.4~40μg/disk and exhibited antimicrobial activity against Streptomyces griseus at 40μg/disk; compounds 2 and 3 showed almost the same activities. Moreover, RK-1009 (1) enhanced the in vitro threonine-phosphorylation of a 60-kDa protein in S. griseus, and its mode of action was distinct from that of well-known kinase or phosphatase inhibitors, proving factumycin-type antibiotics to be a very useful tool for investigating the cellular responses in S. griseus.