The antibacterial activity of fourteen identified compounds from secondary metabolites of fungus Cephalosporium sp. AL031 against eleven gram-positive or gram-negative bacteria in vitro was investigated by agar diffusion assay. The results suggested that two new compounds [S-(+)-5-hydroxymellein, S-(+)-7-hydroxymellein], together with four known compounds [S-(+)-mellein, 2,4-dihydroxy-3,6-dimethylbenzoic acid, 3,6-dihydroxy-2,4-dimethylbenzoic acid and succinic acid] had broad spectrum antibacterial activity. The minimum inhibitory concentrations (MIC) of these six compounds against Shigella sonnei, Mycobacterium tuberculosis and Streptococcus pneumoniae were determined. Their MIC were 0.03125 to approximately 0.5 mg/ml.