Because of the emergence of antibiotic resistance, we must investigate new antibiotical agents. The present study was designed to find new compounds with antibacterial activity from metabolites of Bacillus pumilus. We found that the concentrated fermentation broth of Bacillus pumilus has antibacterial property. By high-performance liquid chromatography (HPLC), three compounds with antibacterial activity were first isolated from the ethyl acetate layer of fermentation broth of Bacillus pumilus. And then their structures were identified by nuclear magnetic resonance (NMR) and mass spectrometry (MS) analysis. According to the data, the compound 1, compound 2, and compound 3 were determined to be 3,4-dipentylhexane-2,5-diol, 1,1'-(4,5-dibutylcyclohexane-1,2-diyl)bis(ethan-1-ol), and 1,1'-(4,5-dibutyl-3,6-dimethylcyclohexane-1,2-diyl)bis(ethan-1-one). And all of them exhibited potent inhibitory effects against a panel of pathogenic bacteria including Staphylococcus aureus ATCC6538, Micrococcus luteus CMCC28001, Variant Salmonella gallinarum CVCC79207, Pasteurella multocida CVCC474, Swine Salmonella, Salmonella enterica ATCC13076, Swine Escherichia coli K88, Chicken Escherichia coli O78. Given its antibacterial activity, 3,4-dipentylhexane-2,5-diol, 1,1'-(4,5-dibutylcyclohexane-1,2-diyl)bis(ethan-1-ol), 1,1'-(4,5-dibutyl-3,6-dimethylcyclohexane-1,2-diyl)bis(ethan-1-one) are assumed to be promising agents for further development as antibacterial agents.