Cutaneous Leishmaniasis (CL) is a vector-borne disease caused by Leishmania species and transmitted by infected female sand flies. CL is widely distributed in Brazil, but knowledge about vectors and transmission cycles could be complex according to localities. The sand fly fauna in Rondônia State is extensive, diverse, and largely unexplored. Although the state records a mean of 1,000 CL cases per year, the vectors of CL are unknown. The aim of this study was to assess phlebotomine sand fly fauna composition using diversity indexes (Shannon [H'] and Simpson [1/D]) and to detect the prevalence of Leishmania infection to verify potential vectors in three ecotopes: (i) forest fragment (FF), (ii) forest edge (FE), and (iii) peridomicile (PE). Captures were performed in four rural districts in the municipality of Porto Velho. A total of 7,026 specimens were captured comprising 72 species, and individuals classified in subgenus level. Overall, the most abundant species were Lutzomyia davisi (n: 1,105), Lutzomyia melloi (n: 760), Lutzomyia auraensis (n: 738) and Lutzomyia antunesi (n: 479). Fauna was most diverse in the FF ecotope (H' = 20.2, 1/D = 11.2), followed by the FE (H' = 18.0, 1/D = 10.1) and PE (H' = 16.6, 1/D = 10.1) ecotopes. Leishmania DNA was detected in 24 of 232 pools. In every ecotope, Leishmania naiffi DNA was identified in the following sand fly species: Lu. antunesi, Lu. davisi, Lu. hirsuta hirsuta, Lu. shawi, Lu. sordellii and Lu. (Trichophoromyia) spp. This observation may indicate that a Le. naiffi transmission focus is present in the study localities. In addition, Leishmania lainsoni was detected in Lutzomyia (Trichophoromyia) spp. Our findings show that sand fly fauna in the study localities is diverse, that Leishmania parasites are circulating in all three ecotopes, and that some sand fly species may be implicated in the transmission of Leishmania to humans in localities evaluated.