Abstract: Despite the availability of modern medicine, number of new infections and resistant bacterial strains is escalating. As a large number of soil bacteria are currently unknown, they represent a promising source for new bioactive natural products. Bacillus sp. 1A1 was isolated from a soil sample, identified, cultivated and its crude extract and purified fractions were evaluated for their potent antibacterial activities. It was identified based on morphological, biochemical and physiological characteristics as well as on molecular level. The 16S rRNA revealed that Bacillus sp. 1A1 is closely related to B. amyloliquefaciens. Its crude extract exhibited inhibiting activity against Gram-positive bacteria. Partial purification of the produced crude extract by size exclusion chromatography yielded three antibacterial fractions that caused inhibition zones of 10-20 mm in agar diffusion assay. Fraction S3 was the most promising fraction that inhibited the growth of tested Gram-positive bacteria with Minimum Inhibitory Concentration (MIC) 31-125 μg mL-1.