This study aims to investigate the secondary metabolites of Streptomyces sp. A5 derived from Sayram Lake were. Five compounds were isolated from the solid fermentation extract of Streptomyces sp. A5 by using various chromatography methods including silica gel column, Sephadex LH-20 column and semipreparative HPLC. Their structures were determined by NMR, MS methods as a new dehydroaltenusin derivative, namely (±)-4'-methoxydehydroaltenusin (1), and four known compounds, including alternariol 4-methyl ether (2), 6,8-dihydroxy-3-methylisocoumarin (3), indole-3-methyl formate (4), streptcytosine H (5). The antibacterial activities of compounds 1-5 were evaluated. The results indicated that compound 1 exhibited moderate antibacterial activity against Escherichia coli, Staphylococcus aureus and Candida albicans with minimal inhibitory concentration (MIC) at 12.5, 6.25 and 6.25 µg/mL, respectively. Compound 2 showed a weaker antibacterial activity against them with same MIC at 25 µg/mL.