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Bacteriophages or phages are specific viruses that are capable of infecting bacteriawithout harming eukaryotic cells. Mycobacteriophages are a specific type of phage that only infectsbacteria in the genusMycobacterium. Mycobacteriophages have been studied for their potentialin killing virulent mycobacteria, such as the etiological agents of tuberculosis and leprosy. In thisstudy, we usedMycobacterium smegmatismc2155 to isolate a mycobacteriophage from Oklahoma[USA] soil. Mycobacteriophage OKCentral2016 was isolated from soil enrichment obtained at theUniversity of Central Oklahoma. This phage produced transparent plaques and has a morphologyconsistent with theSiphoviridaemorphotype. The phage remained stable at temperatures below55°Cand within the pH range of 6-8. The viral replication cycle took approximately 4 hours tocomplete under standard growing conditions. This phage only infectedM. smegmatisand decreasedbiofilm formation in planktonic cultures.