Our objective was to study and evaluate the richness and diversity of Phlebotominae fauna in the Duas Bocas Biological Reserve (DBBR) in the state of Espírito Santo, in southeastern Brazil. Sand fly collections were carried out during four consecutive nights each month between August 2007 and July 2008 at DBBR by using CDC automatic light traps and an illuminated Shannon trap. Specific richness (S) and Shannon diversity index (H) was calculated for each trap. We collected 18,868 sand flies belonging to 29 species and 13 genera. Nyssomyia yuilli yuilli was the most abundant species followed by Psychodopygus ayrozai, Ps. hirsutus, Psathyromyia pascalei, and Ps. matosi. We recorded Brumptomyia cardosoi, Br. troglodytes, and Ps. geniculatus for the first time in the state of Espírito Santo. We discuss the differences in diversity and richness of the sand flies in both traps and in relation to other Brazilian localities and biomes. We also discuss the possibility of wild transmission of Leishmania in the DBBR and the influence of the sand fly species in leishmaniasis transmission to the adjacent areas of the reserve.