The genus Mycobacterium comprises a broad spectrum of obligate microorganisms most notably the M. tuberculosis complex bacteria; potentially-pathogenic or opportunistic species gathered under the common name of M. avium-intracellulare complex, and finally saprophytic mycobacteria. In cattle farming, Mycobacterium bovis (M. bovis) is considered as the principle cause for bovine tuberculosis (bTB). Nevertheless, non-tuberculosis mycobacteria (NTM) are potential pathogens that are increasingly reported to interfere with contemporary intradermal tuberculin tests in veterinary medicine. Therefore, these neglected mycobacteria are capable to make hurdles for bTB diagnosis. In the Iranian environment, the extent and importance of this phenomenon in regard with the public health and economical aspects is just underestimated. This work was therefore intended to isolate and characterize the mycobacterial organisms potentially involved in causing positive reactions in tuberculination of Iranian cattle. Abattoir meat inspection and laboratory microbiological investigation of slaughtered heifers from a 2,000-head Simental cattle farm with 10% of the herd positive tuberculin reactors, failed to provide any evidence of M. bovis infection. No mycobacteria was detected in bacterial culture of specimens from feedstuff, water or even the visiting feral rodents of the farm. When the farm workers were included in assessments, 22 individuals were found tuberculin-positive with 5 of them also smear-positive in their sputa assessment. Molecular identification of the five isolates from workers through sequencing of the 16SrRNA proved their identity as Mycobacterium thermoresistibile (Mth). Removing of the carrier workers from the farm vicinity, successfully ended the issue of the constantly-positive tuberculin test in the herd with no return of the problem until now. We are convinced further investigations are essential in search for interference extent and importance of NTM species in cattle tuberculination scheme in Iran.