Fruit flies are a familiar sight in many food service facilities. Although they have been long considered as “nuisance pests,” some of their typical daily activities suggest they may pose a potential public health threat. The aim of this study was to provide evidence of the ability of small flies to transfer bacteria from a contaminated source, food, or waste to surfaces or ready-to-eat food. Laboratory experiments were conducted by using purpose-built fly enclosures to assess the bacterial transfer capability of fruit flies. Drosophila repleta were capable of transferring Escherichia coli O157:H7, Salmonella Saint Paul, and Listeria innocua from an inoculated food source to the surface of laboratory enclosures. In addition, using an inoculated doughnut and noncontaminated lettuce and doughnut surfaces, fly-mediated cross-contamination of ready-to-eat food was demonstrated. Fruit flies were shown to be capable of accumulating approximately 2.9 × 103 log CFU of E. coli per fly within 2 h of exposure to a contaminated food source. These levels of bacteria did not decrease over an observation period of 48 h. Scanning electron micrographs were taken of bacteria associated with fly food and contact body parts and hairs during a selection of these experiments. These data, coupled with the feeding and breeding behavior of fruit flies in unsanitary areas of the kitchen and their propensity to land and rest on food preparation surfaces and equipment, indicate a possible role for fruit flies in the spread of foodborne pathogens.