The Mycobacteria are a genus of bacteria which are acid-fast, non-motile, grampositive rods. The genus comprises several species classified into three main groups. Firstly, the major group of these organisms, which poses the biggest threat, is the M. tuberculosis complex which can cause tuberculosis-like disease. These include M. bovis, M. africanum and M. microti. Members of the M. tuberculosis complex are not found in the environment. The second group is M. leprae which is the causative agent of leprosy. The last group constitutes the nontuberculous mycobacteria (NTM), which are all the environmental mycobacteria that can cause various diseases resembling tuberculosis. Due to the importance of environmental mycobacteria, 15 mycobacteria isolates were isolated from environmental samples such as soil, water and drinking water biofilms. After PCR amplification of the hsp65 gene using genus specific primers hsp65, the isolates revealed sequences similarities when compared with the well characterized mycobacteria in the GenBank. Alignment of the nucleotide sequences and homology analysis were done with Clustall. It has been suggested that mycobacteria-associated phages (mycobacteriophages) may make an important contribution to the evolution of pathogenic mycobacteria. Spontaneous induction of phage associated with mycobacteria isolates using overlay and indicator plate methods was not successful to detect the presence of any inducible phage. A phage was isolated from soil samples that was designated the name A22. After purification and characterization. A22 phage was compared morphologically to well characterized L5 phage using electron microscopy. Morphological studies revealed that A22 mycobacteriophage had a non-contractile tail approximately 150 nm long with an isometric head approximately 60 nm, the phage could be assigned to the family Siphoviridae, According to these criteria, both of the phages (A22 and L5) belong to the order Caudovirales (tailed bacteriophages). Based on PCR amplification of A22 phage DNA using L5 gp71 specific primers and the infection of M. smegmatis L5 lysogen, we believe that this novel A22 phage differs from L5 phage.