Abstract For the definitive diagnosis of bovine tuberculosis, isolation of the etiologic agent is required. However, there is no consensus on the best methodology for isolation of Mycobacterium bovis in Brazil. This study evaluated the most used decontaminants and culture media in the country, in order to identify the best combination for the Brazilian samples. Three decontaminants - 2% sodium hydroxide (w/v), 0.75% hexadecylpiridinium chloride (w/v) and 5% sulphuric acid (v/v) and four culture media - 7H11 Middlebrook with additives and OADC supplement “A” (7H11 A), the same media with another supplement trademark (7H11 B), tuberculosis blood agar (B83) and Stonebrink's medium were compared. Regarding the isolation, there were no significant differences between the decontaminants and media combinations, except 7H11A combined to any decontaminant. However, the mean colonies score was significantly greater when the samples were decontaminated with 5% sulphuric acid and inoculated in 7H11 B or SB, without significant difference between them, although colonies appeared earlier on 7H11B than on SB. The trademark of OADC supplement influenced the isolation rate and the number of isolated colonies in Middlebrook 7H11. An incubation time of four weeks was required to detect all positive samples in 7H11 B after decontamination with 5% sulphuric acid but there was an increase in the number of colonies until the sixth week of incubation. Overall, the best strategy for the primary isolation of M. bovis from Brazilian samples was the decontamination with 5% sulphuric acid (final concentration) and inoculation in Middlebrook 7H11 medium formulated with OADC supplement “B”.