We evaluated prevalence and distribution of antibiotic resistant E. coli in three categories: animals (goats, pigs, poultry, cattle, sheep), humans (butchers, meat sellers, animal farm workers, buyers) and animal food products (milk, cheese, beef, chicken, yoghurt) from selected animal farms in South-West Nigeria. The biofilm formation, phylogrouping and detection of biofilm-associated genes were further analyzed. Out of a total number of 280 samples that were collected, 216 E. coli strains were isolated. The prevalence of isolating E. coli from humans (96%) was higher than from animals (89%) and about 38.8% were isolated from animal food products. Out of these 216 E. coli isolates that were tested for antibiotic sensitivity, 37 were multidrug-resistant. The prevalence of isolating these multi-resistant strains was highest in the animal category (23.6%) followed by the animal food products (16.1%) and the human category (11.5%). All the multidrug-resistant isolates were capable of forming biofilm, which varied significantly with each isolate. The majority of isolates belonged to phylogroup B1. All biofilm-associated genes were detected in the antibiotic resistant isolates. The results suggest the need for continuous surveillance of antibiotic resistance, biofilm formation and virulence determinants in E. coli in the whole food chain.