The transmission of antibiotic resistance (AR) from farm animals to healthy human communities, beyond the food chain, is often facilitated by biological vectors, notably houseflies (Musca domestica). This study aimed to evaluate the role of M. domestica collected from commercial broiler chicken farms as a carrier of multidrug-resistant (MDR), extended-spectrum β-lactamase (ESBL)-producing Escherichia coli. E. coli were isolated separately from the housefly's external surface (ES) and internal homogenate (IH) to determine the primary AR transmission route within houseflies. Remarkably, 68.6% houseflies harboured E. coli. Isolated E. coli were evaluated for susceptibility to clinically relevant antibiotics and screened for the presence of 22 plasmid-borne AR genes (ARGs) using PCR. Results revealed significant resistance to key antibiotics, with >
70% of isolates resistant to ampicillin and >
50% resistant to tetracycline and nalidixic acid in both ES- and IH-derived E. coli. Notably, a significant prevalence of resistance was observed to third-generation cephalosporins. Additionally, >
80% of E. coli isolates were MDR. A statistically significant difference (unpaired t-test, p <
0.05) was observed in the presence of ESBL-producing E. coli between the houseflies' ES (28.14%) and IH (38.14%). ARGs such as, ampC, tetA, qnrS, strA, strB, and sul3 were frequently detected in both ES- and IH-derived E. coli isolates. Among the ESBL-producing genes, bla