This research article addresses the hypothesis that vehicles used for cattle transport are contaminated with Escherichia coli, a potential foodborne pathogen, despite current regulations on sanitation practices. Dairy cattle and calves are regularly transported to auction markets, calf rearers and slaughterhouses. UK Government guidelines require livestock transport vehicles to be cleaned and disinfected within 24 hours of use or before re-use within that period. It is feasible, however, that if cleaning fails to eradicate bacteria, then transport vehicles can act as a fomite in the spread of antimicrobial resistance (AMR) pathogens. In this study, 13 trailer-loads (TLs) of calves were transported for 40–60 minutes. Trailers were then cleaned and disinfected within 20 minutes of unloading. Five sites within the trailer were swabbed after pressure washing and again 30 minutes after application of disinfectant. A bacterial count for E. coli was performed through growth on selective agar, and species identification was confirmed by MALDI-TOF. A subset of 30 isolates was selected for antibiotic susceptibility screening to a panel of veterinary and human antibiotics. E. coli were recovered from all TLs and sites; however, not all sites were contaminated in each TL. E. coli count was significantly reduced, but not eliminated, following application of disinfectant. Furthermore, high prevalence of resistance to sulphonamides, first-generation cephalosporins, and tetracyclines was observed. Forty percent of screened isolates were also classified as multidrug-resistant (MDR) (i.e. resistant to at least one antibiotic from three or more antibiotic classes). Application of disinfectant did not increase the risk of recovering an MDR isolate. This study demonstrates that livestock trailers can harbour potential zoonotic pathogens with AMR properties. Disinfection in accordance with current guidelines is an important step in reducing, but not eradicating, bacterial populations in these vehicles. Improved cleaning and/or disinfection policies are required to mitigate the potential for AMR transmission.