House flies, Musca domestica L., develop within and feed upon microbe-rich substrates such as manure, acquiring and potentially disseminating pathogenic bacteria. Because adult female flies frequent manure due to oviposition or nutrition requirements, we hypothesized females would consume more manure than males even in the presence of additional food sources (e.g. sugar), resulting in measurable differences in bacterial load between sexes. House fly acquisition of bacteria from manure inoculated with GFP-expressing E. coli or Salmonella sp. was examined for both sexes over 24 h in assays where 1) inoculated manure was the only food source and 2) both inoculated manure and sugar water were provided. We conducted assays with mated male and female flies separately to determine sex-specific effects on bacterial acquisition. Over 24 h, bacterial abundance increased in manure inoculated with S. Typhimurium, but not E. coli. In flies, bacterial abundance increased within sex only in S. Typhimurium assays. Overall, female flies harbored more bacteria than males; however, differences in abundance were only significant at early time points. In the E. coli manure–sugar assays, male and female colony-forming units (CFU) abundance differed at 4 and 12 h, while CFU abundance differed at 4 and 12 h in all S. Typhimurium assays. Fly digestive tract observations from manure–sugar assays supported these initial differences especially at 4 h where females contained manure and fly food, while males contained only sugar water. Identifying sex-specific effects on house fly acquisition and carriage of bacteria from manure facilitates risk assessment of pathogen transmission on farms.