BACKGROUND: Phlebotomine sand flies (Diptera: Psychodidae) are insects of medical importance due to their involvement in the zoonotic transmission of Leishmania spp. to vertebrates. The aim of this work was to study the ecology of the sand fly fauna of two types of environments, a rural environment (the Transacreana Road) and an urban park (Horto Florestal Park), both located in the municipality of Rio Branco in the state of Acre, Brazil. Additionally, this study intended to investigate Leishmania infection and blood meal sources of these sand flies using molecular techniques. METHODS: The sand fly fauna was studied in different environments (i.e. forest and peridomestic environments in a rural area, and an urban forest) using Shannon traps and HP light traps to collect sand fly specimens over 13 consecutive months (December 2014 to January 2016). For investigating natural infection by Leishmania and the source of sand fly blood meals, DNA samples were extracted from female sand flies and subjected to polymerase chain reaction targeting ITS1 and cytb genes. DNA sequencing was subsequently used to identify species of Leishmania and the source of blood meals. RESULTS: A total of 2515 individual sand flies of 43 species were collected and identified, Trichophoromyia auraensis (839; 33.35%), Trichophoromyia spp. (537; 21.35%) and Evandromyia saulensis (187; 7.43%) were more abundant in the rural area (S = 41 species) than in the urban forest. No significant differences were found in species richness between forest and peridomestic environments in the rural area (H = 0.04; P > 0.05), but a larger number of species was found in the forest. Leishmania DNA was sequenced in 13 samples, confirming the presence of L. (V.) braziliensis in Th. auraensis (n = 1), Ev. saulensis (n = 2), Ev. walkeri (n = 1), Ps. llanosmartinsi (n = 1), Pi. nevesi (n = 2), Ps. davisi (n = 1), Ps. ayrozai (n = 1), Pa. aragaoi (n = 1), Ny. antunesi (n = 1) and Ev. infraspinosa (n = 1). Only Ps. ayrozai possessed a sequence similar to that of L. (V.) guyanensis (99%). Through microscopic analysis, five specimens of Ev. saulensis were found to possess flagellate forms in the hindgut, with an infection rate of 2.4%. Samples from 33 fed females were submitted to cytb gene amplification, for which sequencing determined that all were similar to the sequence deposited on GenBank for Gallus gallus (domestic chicken). CONCLUSIONS: The high abundance of Trichophoromyia auraensis and Ev. saulensis, and the detection of L. (V.) braziliensis DNA, suggests that both species may be vectors of American tegumentary leishmaniasis. Psychodopygus ayrozai was found to be infected by L. (V) braziliesnsis and L. (V.) guyanensis, and although collected in low abundance, it may be a potential vector in the region. The sand fly fauna was found to be rich and diverse with predominance of the genus Psychodopygus. Identification of food sources of fed females showed that 100% amplified a gene region compatible with the domestic chicken, which although considered refractory in the disease transmission cycle, may have an influence on the population dynamics of sand flies.