Therapeutic failure in a human immunodeficiency virus-negative patient with visceral leishmaniasis was due to mixed infection by two different Leishmania infantum zymodemes: L. infantum zymodeme MON-98, which is a rare zymodeme and is reported for the first time in Greece, and zymodeme MON-1, which is common in the Mediterranean region. The two strains were isolated from two samples of bone marrow from the patient obtained before the administration of treatment and 20 days later, since there was no improvement in the clinical signs. The zymodemes MON-98 and MON-1 exhibited different behaviors in vitro and showed different sensitivities to meglumine antimoniate in vitro and in vivo, as shown by clinical findings. Mixed infections with different Leishmania strains may explain the differences in the clinical course of leishmaniasis in many patients and may be the reason for treatment failures.