The culture of Mycobacterium avium subspecies paratuberculosis (Map) is considered as the "Gold Standard" diagnostic test for paratuberculosis. On account of the high contamination rates with an average of 30% observed after conventional fecal culture from beef cattle herds, we address this problem by identification of Pseudomona aueruginosa as the most common contaminant. After antibiotics susceptibility test the following antibiotics were efficient controlling the grow of pseudomona: enrofloxacin, florfenicol and gentamicin. To check the in vitro breakpoint inhibition effect of these antimicrobial agents to Map, clinical isolates of paratuberculosis agent were tested to examine their growth responses in Herrold´s medium supplemented with these antibiotics at serial dilutions. Inhibition effect on Map was observed in enrofloxacin and gentamicin at low concentration. Florfenicol combined the best in vitro breakpoint inhibition effect on contaminant grow with minimal susceptibility to clinical isolates of Map.