BACKGROUND: Zoonotic Cutaneous Leishmaniasis (ZCL) is a polymorphic disease. It is generally accepted that bacterial superinfection may play a role in the clinical appearance of the lesions and may delay or prevent the healing process. However, the pattern of bacterial pathogens involved has rarely been investigated. MATERIAL/METHODS: The aim of this study was to identify the bacterial species contaminating the suspected ZCL and their susceptibility to commonly used antibiotics. Microscopic examination of stained smears and cultures were used to differentiate ZCL from non-ZCL lesions in a rural area north of Isfahan, Iran from July to December 2009. Bacteria were isolated from the lesions and identified and antibiotic susceptibility was determined by standard microbiological techniques. RESULTS: The results show that 602 (68%) of 855 patients were positive for ZCL, of which 83.4% with volcano-shape, 8.8% psoriasiform, 6.6% popular form and 1.2% with other atypical forms of ZCL. The bacteria were isolated from 66.8% of ZCL (70% of volcano-shape, 60% of psoriasiform and 25% of popular form) and 64.7% of non-ZCL lesions. The most common species were Staphylococcus aureus (41.7%) and S. epidermidis (28%) followed by Bacillus sp. Streptococcus pyogenes, Escherichia coli, Klebsiella sp., Proteus sp., Enterobacter sp. and Pseudomonas aeroginosa. Ciprofloxacin, Erythromycin, Cefazolin and Clindamycin were the most effective antibiotics. CONCLUSIONS: Bacterial superinfection appears to be very common in ZCL, but its prevalence is not different from that of non-ZCL lesions and it has little effect on the clinical appearance of anthroponotic cutaneous Leishmaniasis (ACL). Local lesion care and management of bacterial superinfection must be considered in the treatment of ZCL.