This study aimed to assess the influence of the presence of synanthropic flies in food preparation environments on the transmission of potentially zoonotic gastrointestinal protozoa. Flies were captured using a glass containing water, fruits, and pieces of protein. Approximately 260 flies from four different species were captured: Musca domestica (76.92%; 200/260), Cochliomyia hominivorax (17.31%; 45/260), Lucilia cuprina (3.85%; 10/260), and Hydrotaea aenescens (1.92%; 5/260). Protozoa were identified through microscopic analysis after macerating these arthropods contained in their respective eppendorfs, using two techniques, namely Mini-FLOTAC® and centrifugal sedimentation with Ziehl-Neelsen staining. The analysis revealed a concerning scenario regarding the epidemiological chain of gastrointestinal protozoa in Sergipe, Northeast Brazil. The positivity rate was 26.92% (14/52) of the pools, with 100.00% (5/5) in Aracaju, 30.00% (3/10) in Nossa Senhora do Socorro, 27.27% (3/11) in Nossa Senhora da Glória, 12.50% (2/16) in Carmópolis, and 10.00% (1/10) in Nossa Senhora das Dores. Two species of protozoa were identified, namely Cryptosporidium spp. (23.08%; 12/52) and Entamoeba spp. (9.62%; 5/52). The involvement of two fly species in the maintenance of the life cycle of these protozoa was noticeable, specifically M. domestica (27.50%; 11/40) and C. hominivorax (22.22%; 2/9). Counties analyzed had an human development index (HDI) considered medium; however, they still faced socioeconomic problems such as absence of sanitation systems, waste accumulation in the streets, and illegal waste disposal, which could favor the proliferation of these vectors and the spread of these gastrointestinal protozoa.