Vanessa, Eliana, Simone, Cris Brito, Tati, Leslie, Iara, Luís Ivan, Maurício, Letticie, Adriano, Ricardo, Richard, Alexandre, Silvio, Renata Paixão, Sandra, Danival, Cristina, Virginia, Bispo isolation of mycobacteria in milk samples was practicable against the tradicional one; 2) the time needed for the detection of mycobacteria colonies was slightly less with the thin layer technique than the tradicional culture method; 3) techniques 1 and 2 gave more number of colonies and more proportion of positive culture than the tradicional one in all media used; 4) this technique should be used as a complementary method to the tradicional one in the diagnostic of bovine tuberculosis from milk samples in orther to improve epidemiologic vigilance.