Patients with pulmonary tuberculosis (TB) are the most important source for TB infection, being the risk of infection determined by the source case infectiousness and the contact closeness. Currently, the administration of isoniazid is used to prevent the infection to some extent in household contacts. At experimental level, defensins are efficient molecules for the treatment of TB and other infectious diseases. In this work, we used a model of Mycobacterium tuberculosis transmission by long cohabitation of infected and noninfected mice, and treated the latter group with antimicrobial peptides in order to determine the potential capacity of defensins to prevent the infection. Our results showed that the intratracheal administration of human neutrophil peptide-1, human β-defensin-2 alone or in combination and the use of L-isoleucine significantly prevents bacterial transmission, diminishing pulmonary lesions and bacterial loads. Data suggest the potential use of L-isoleucine as prophylactic for TB household contacts.