Phlebotomine sand flies are worldwide vectors of Leishmania parasites as well as other bacterial and viral pathogens. Due to the variable impact of traditional vector control practices, a more ecologically based approach is needed. The goal of this study was to isolate bacteria from the most attractive substrate to gravid Phlebotomus papatasi Scopoli sand flies and determine the role of bacterial volatiles in the oviposition attractancy of P. papatasi using behavioral assays. We hypothesized that gravid sand flies are attracted to bacterially derived semiochemical cues associated with breeding sites. Bacteria were isolated from a larvae-conditioned rearing medium, previously shown to be highly attractive to sand flies. The isolated bacteria were identified by amplifying and sequencing 16S rDNA gene fragments, and 12 distinct bacterial species were selected for two-choice olfactometer bioassays. The mix of 12 bacterial isolates elicited strong attraction at the lower concentration of 10⁷ cells per ml and significant repellence at a high concentration of 10⁹ cells per ml. Three individual isolates (SSI-2, SSI-9, and SSI-11) were particularly attractive at low doses. In general, we observed dose-related effects, with some bacterial isolates stimulating negative and some positive dose–response curves in sand fly attraction. Our study confirms the important role of saprophytic bacteria, gut bacteria, or both, in guiding the oviposition-site selection behavior of sand flies. Identifying the specific attractive semiochemical cues that they produce could lead to development of an attractive lure for surveillance and control of sand flies.