Mycobacteriophages are viruses that infect Mycobacterium spp. Till date, 10427 mycobacteriophages have been isolated and 1670 mycobacteriophage genomes have been sequenced https://phagesdb.org/hosts/genera/1/ (cited on 30th December,2018). In the previous study, 10 different mycobacteriophages from 14 soil samples were isolated, by qualitative plaque formation method using Mycobacterium smegmatis as host. Among these, three phages were found to infect four different species of Mycobacterium, i.e., Mycobacterium fortuitum subsp. fortuitum MTCC993, Mycobacterium kansasii MTCC3058, Mycobacterium avium subsp. avium MTCC1723, and Mycobacterium tuberculosis MTCC300, besides the host M. smegmatis. The phage lysates were concentrated by polyethylene glycol (PEG) precipitation. One of the three phages showing host diversity was selected for further study. The various phage growth parameters such as incubation temperature, time of adsorption, host cell density and effect of cations were standardised.The studies were done by qualitative and quantitative plaque assay method.The phage selected for further study showed an optimum adsorption time of 15 min. The optimum temperature for propagation was found to be 37°C. The phage was found to be stable at 42°C. In the presence of calcium, the phage showed a higher rate of infectivity.Understanding the biology of mycobacteriophages and their host diversity is the key to understanding mycobacterial systems. This could be the first step toward exploiting the potential of phages as therapeutic agents.