Filth flies play a major role in the transmission of microbial organisms that cause disease in animals and humans. A procedure was developed using filter paper to collect filth fly fecal/regurgitation droplets at dairies and turkey production facilities that could be used to detect pathogen DNA carried by filth flies. Weekly fly fecal/regurgitation droplets were collected on 11 cm diameter filter paper that was either tacked to beams or stapled to 30 cm wooden stakes. Molecular diagnostics using polymerase chain reaction (PCR) procedures detected the presence of Escherichia coli H7, Campylobacter sp., and Cochlosoma anatis DNA in fecal/regurgitation droplets deposited by filth flies at two dairy and one turkey farm in Arkansas. This procedure provided a rapid and effective method to monitor pathogen presence in filth fly populations.