Brain ischemia and subsequent reperfusion injury is a serious disorder, ranking as the third leading cause of death and most common cause of adult disability in Japan. L-glutamate-induced neuronal cell death is a key mechanism of brain ischemic injury. In the course of screening for inhibitors of glutamate toxicity using the neuronal hybridoma N18-RE-105 cells as an in vitro ischemia model, novel compounds designated aestivophoenins A and B were isolated from Streptomyces purpeofuscus 2887-SVS2. The producing organism was cultivated in a fermenter, and the compounds were purified via mycelial acetone extraction, ethyl acetate partitioning, silica gel column chromatography, ODS column chromatography, and HPLC. Their structures were elucidated by high-resolution FAB-MS, UV, IR, and NMR (DQF COSY, HMBC) analyses: aestivophoenin A (C31H32N2O7) and B (C36H40N2O7) are benzoated phenazine derivatives with a rhamnose moiety, and B is an isoprenyl adduct of A. Both compounds effectively suppressed L-glutamate toxicity in N18-RE-105 cells with EC50 values of 15.0 nM and 6.2 nM, respectively, and also protected rat embryonic primary hippocampal neurons from glutamate toxicity.