The present study was performed to report the bacterial identification, optimization of culture conditions and characterization of a novel antibiotic substance from a Bacillus sp. EA62 strain isolated from soil. The EA62 strain was identified based on 16S rRNA analysis. The new isolate EBD 9-1 showed 100% sequence identity with Brevibacillus laterosporus. The antibacterial activity of the new substance was examined against five pathogenic bacteria and was observed to be the most effective against Escherichia coli. An agar diffusion assay was performed to evaluate the antibacterial activity of the substance. The effects of some nutritional (amino acid, carbon, nitrogen and metal sources) and physical factors (pH and temperature) and incubation time on the antibiotic activity were studied. Antibiotic activity in basal medium reached the maximum levels after 72 h of incubation. The best antibiotic activity was obtained in the presence of glucose as a carbon source, yeast extract as a nitrogen source, glutamic acid as an amino acid source and MgSO4+CaCO3 as metal ion sources. For physical parameters, the best results were obtained at 37 degrees C, pH 7.0. The antibiotic substance was partially purified, and the estimated molecular weight was 6.3 kDa. The minimum inhibitory concentration (MIC) values determined against five pathogen bacteria were >256 mu g/ml. The substance was identified by thin-layer chromatography, and its Rf value was measured as 0.04 cm.