House flies associate with microbes throughout their life history. Bacteria ingested by adult flies enter the alimentary canal and face a hostile environment including antimicrobial defenses. Because the outcome of this interaction impacts bacterial survival and dissemination, our primary objective was to understand the temporospatial dynamics of fly-bacteria associations. We concurrently examined the temporospatial fate of GFP-expressing Pseudomonas aeruginosa (GFP-P. aeruginosa) in the house fly alimentary canal along with antimicrobial peptide (AMP) expression. Motile, viable GFP-P. aeruginosa were found in all regions of the alimentary canal and were culturable throughout the observation period (2-24 h). A significant decrease in recoverable bacteria occurred between 2 and 12 h, followed by an increase between 12 and 24 h. qRT-PCR analysis showed expression of the AMPs cecropin, diptericin, and defensin both locally (gut) and systemically. Furthermore, mRNA of all AMPs were expressed throughout gut tissues, with some tissue-specific temporal variation. Interestingly, fluctuation in recoverable P. aeruginosa was associated with AMP protein expression in the gut (immunofluorescent signal detection), but not with mRNA (qRTPCR). In regards to vector competence, flies excreted GFP-P. aeruginosa throughout the 24 h period, serving as both reservoirs and disseminators of this bacterium. Collectively, our data show flies can harbor and disseminate P. aeruginosa, and that the interactions of fly defenses with bacteria can influence vector competence.