Aims: This study investigated the antibiotic susceptibilities of avian pathogenic Escherichia coli (APEC) isolates from layer chickens that died of colibacillosis, in the 6 states of Southwest Nigeria. Methodology and results: From bacteriological cultures of dead layers with suspected colibacillosis, 509 APEC isolates were identified by Gram staining, microscopy, and biochemical reactions, and tested by the Kirby-Bauer disc diffusion method for susceptibility to 9 human and veterinary antibiotics: Streptomycin (Str, 200 µg); Nitrofurantoin (Nit, 300 ?g); Augmentin (Aug, 30 µg); Amoxicillin (Amo, 25 µg); Chloramphenicol (Chl, 30 µg); Gentamicin (Gen, 10 µg); Tetracycline (Tet, 25 ?g); Ofloxacin (Ofl, 5 µg); and Fosfotrim (Fos, 30 ?g). The APEC isolates were highly resistance to Amo (99.2%), Chloramphenicol (96.5%), Tet (91.4%) and Ofl (86.4%); moderately resistant to Gen (67.2%), Aug (63.5%), Nit (51.7%) and Fos (37.9%); while 14.3% of the isolates were resistant to Str at the test concentration of 200 µg, equivalent to ten times the normal disc test concentration. Overall, 39 antibiotic resistance patterns were observed, and more than 84.68% (n = 431) of the APEC isolates were multi-drug resistant. Conclusion, significance and impact of study: These results indicate that there is widespread misuse of antimicrobials in the poultry industry in Southwest Nigeria, and portend a high failure rate for the treatment of avian colibacillosis in the region. Owing to the public health importance of multidrug resistant bacteria that arise from animal production systems, there is need for exhaustive review of the regulatory framework guiding the supply and use of veterinary antimicrobials in Nigeria.