Sputum from clinically confirmed patient and lung tissues from tuberculin positive cattle were taken from el Menia governorate. The samples were subjected to mycobacterial isolation on Lowenstein Jensen media. Human samples gave 8 out of 20 positive culture with isolation rate of 40 % where animal samples gave 11 out 15 positive culture with isolation rate of 73.3%. Biochemical identification revealed that all the human isolates belonging to M. tuberculosis where animal isolates where exclusively M. bovis. Molecular characterization using PCR utilizing primers set amplifying IS6110 gene revealed that all human and animal isolated were positive giving amplicon with relative molecular size of 254bp. When using primer set for detecting of the RD8 region, only human isolates gave a clear amplicon migrating about 673 bp which confirms that these isolates were belonging to M. tuberculosis.