Alpacas (Vicugna pacos) are growing in popularity and are increasingly being presented for veterinary care. Literature reports indicate that dermatophytosis occurring in alpacas accounted for about 3% of dermatological diagnoses. However, there are no reports regarding species of dermatophytes associated with alpacas and reservoirs of infection. In this study, we investigate the diagnosis and epidemiological origin procedure and the virulence enzymes activities of Trichophyton benhamiae isolates obtained from alpacas from a breeding farm. Identification was carried out traditionally by correlating clinical manifestations with micro- and macroscopic examination, and molecular differentiation methods based on Internal Transcribed Spacer (ITS) sequences. Epidemiological analysis was carried out on the basis of Melting Point PCR (MP-PCR) and Amplified Fragment Lenght Polymorphism (AFLP) genotyping. The production of virulence factors was evaluated phenotypically using specific test media. The results obtained from diagnostic tests indicated that the etiological factor of dermatophytosis is T. benhamiae. The same species was also isolated from cowsheds and insects. The MP-PCR and AFLP analyses indicated high invariability of the genomes of the strains isolated from the animals, cowsheds, and insects. In conclusion, animal husbandry outside the natural ecological niche may increase predisposition to dermatophytosis. The treatment of animals alone is insufficient, one should be aware that only elimination of all fungal sources is a long-term success and the key point of therapy.