Antimicrobial proteins/peptides have attracted much attention because of their potential use in the industrial setting. In the present study, a thermostable antimicrobial protein (BSAMP) was purified from the culture supernatant of Bacillus subtilis FB123 by ammonium sulfate precipitation, gel chromatography on Sephacryl S-200 High Resolution, and ion exchange chromatography on DEAE Sepharose Fast Flow column. The molecular weight of the purified BSAMP was 54 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis both in the absence and presence of β-mercaptoethanol. Its isoelectric point was determined to be 5.24 by isoelectric focusing electrophoresis. Periodic acid-Schiff staining revealed BSAMP to be a glycoprotein. Maximum activity was obtained at pH 6.0, with over 79% maximum activity retained at pH 3.0-5.0 and pH 7.0-9.0, respectively. BSAMP was shown to be highly thermostable, as its activity did not change obviously after treatment at 100 °C. However, it was partially sensitive to papain, trypsin, and alkali proteases. Finally, the bacterial protein exhibited broad-spectrum antimicrobial activity against several pathogenic organisms. These findings suggested that BSAMP should be further developed as a natural antibacterial agent for disease prevention in aquiculture and agriculture.