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Background: Phlebotomine sand flies are the vectors of the leishmaniases, parasitic diseases caused by Leishmania spp. Little is known about the prevalence and diversity of sand fly microflora colonizing the midgut or the cuticle. Particularly, there is little information on the fungal diversity. This information is important for development of vector control strategies. Methodology/Principal Findings:Five sand fly species: Phlebotomus papatasi, P. sergenti, P. kandelakii, P. perfiliewi and P. halepensis were caught in Bileh Savar and Kaleybar in North-Western Iran that are located in endemic foci of visceral leishmaniasis. A total of 35 specimens were processed. Bacterial and fungal strains were identified by routine microbiological methods. We characterized 39 fungal isolates from the cuticle and/or the midgut. They belong to six different genera including Penicillium (17 isolates), Aspergillus (14), Acremonium (5), Fusarium (1), Geotrichum (1) and Candida (1). We identified 33 Gram-negative bacteria: Serratia marcescens (9 isolates), Enterobacter cloacae (6), Pseudomonas fluorescens (6), Klebsiella ozaenae (4), Acinetobacter sp. (3), Escherichia coli (3), Asaia sp. (1) and Pantoea sp. (1) as well as Gram-positive bacteria Bacillus subtilis (5) and Micrococcus luteus (5) in 10 isolates. Conclusion/Significance:Our study provides new data on the microbiotic diversity of field-collected sand flies and for the first time, evidence of the presence of Asaia sp. in sand flies. We have also found a link between physiological stages (unfed, fresh fed, semi gravid and gravid) of sand flies and number of bacteria that they carry. Interestingly Pantoea sp. and Klebsiella ozaenae have been isolated in Old World sand fly species. The presence of latter species on sand fly cuticle and in the female midgut suggests a role for this arthropod in dissemination of these pathogenic bacteria in endemic areas. Further experiments are required to clearly delineate the vectorial role (passive or active) of sand flies. [ABSTRACT FROM AUTHOR]