Phenazines are a large group of natural and synthesized nitrogencontaining heterocycles, including more than 100 different compounds of natural origin.1 The group of phenazine compounds exhibit a broad range of biological activities, such as antibacterial, antimalarial, antitumor and antiparasitic activities.2 Natural phenazines are mainly isolated from different microorganisms, including Pseudomonas, Streptomyces and miscellaneous genera.2 Even though large numbers of phenazines have been isolated from natural sources,1,3–6 the dimeric phenazines were relatively rare. Only esmeraldines A–B,7 phenazostatins A–D,8–11 izumiphenazines A–B12 and phenazinolines A–E13 were found till now and all of them were isolated from streptomycete. In the course of screening for new bioactive compounds from endophytic actinomycete sources, a new cytotoxic dimeric phenazine, diastaphenazine (1), along with a known compound, izumiphenazine C (2) (Figure 1) was isolated from the fermentation broth of Streptomyces diastaticus subsp. ardesiacus. In this paper, we report the fermentation, isolation, structural elucidation, and cytotoxic and antibacterial activities of 1.