Increasing antimicrobial resistance (AMR) colonizing domesticated animals is a global concern threatening food safety. This study aimed at determining the prevalence of multidrug resistance (MDR) and epidemiological cut-off values (ECVs) of E. coli isolated from poultry and pigs in Mwanza, Tanzania. This cross-sectional study was conducted between June and August 2021, involving 297 pigs, 191 broilers, and 203 layers. Rectal and cloacal swabs were collected and processed following standard guidelines. ECVs were determined using normalized resistance interpretation (NRI), a computer software, and descriptive analysis was performed using STATA version 13.0. The overall prevalence of MDR E.coli was 63.2%, whereas poultry (87.5% layers and 86.3% broilers) were more colonized than pigs (31.8%) (p < 0.001). Based on ECVs of antibiotics tested, E. coli from broilers, layers, and pigs exhibited different resistance patterns hence different populations. Exotic breed (p < 0.001) and recent antimicrobial use (p < 0.001) significantly predicted colonization with MDR E. coli. Veterinary officers should implement regulations that prohibit the inappropriate use of antimicrobial agents in livestock keeping.