BACTERIOCINS are considered as ideal candidates for several health care applications due to their limited range of activity and rapid degradability by proteolytic enzymes. Eight bacteriocin-producing Bacillus amyloliquefaciens isolates were screened by polymerase chain reaction (PCR) using four sets of primers designed specifically to detect bacteriocin-producing genes on their chromosomes. Gene encoding for Amylocyclicin was detected in four isolates. A phylogenetic data analysis of the four Amylocyclicin-predicted proteins placed them in a separate node with their closest relatives, B. amyloliquefaciens and Bacillus velezensis strain FZmhtB, which until recently, was a member of the B. amyloliquefaciens species. Surprisingly, Subtilosin producing gene was detected in two of the previously mentioned isolates indicating that they contain multiple bacteriocin encoding genes, an unusual phenomenon for Bacillus amyloliquefaciens isolates. The remaining four isolates lacked any known bacteriocin gene family and are anticipated to contain novel gene types. The most potent of these four isolates was chosen for further large-scale production and extraction of its bacteriocin. Antibacterial activity of the extracted bacteriocin was detected in the protein fraction under the membrane cut-off value of <10,000kDa against gram-negative and gram-positive indicator bacterial isolates, with a larger average inhibition zone diameter observed for the gram-positive isolate. Furthermore, SDS-PAGE analysis of the partially purified active bacteriocin fraction revealed a protein fragment with a relative molecular weight between 7 and 7.5kDa. The PCR assay in this study provided coverage for all known B. amyloliquefaciens bacteriocins allowing the quick and easy screening for the presence of bacteriocin-encoding genes.