Background: Colibacillosis caused by avian pathogenic Escherichia coli (APEC), which is an extraintestinal pathogenic E. coli (ExPEC), is the commonest bacterial disease of poultry, which has led to severe economic losses. Methods: In this study, we isolated ten suspected strains of Escherichia coli (E. coli) from the droppings of birds suffering from colibacillosis at the teaching and research farm of the Niger Delta University, Wilberforce Island, Nigeria. Eight virulence associated genes (VAGs) were analysed for, using multiplex PCR techniques. Results: Only four (Isolates 1, 5, 6 and 8) were positive for the tested virulent genes. The iron uptake gene iutA, was predominant, being present in all the four isolates (1, 5, 6 and 8). Another iron uptake gene iucD, was detected only in isolates 1 and 5, while outer membrane protein gene ompT, was detected in isolates 1 and 8 only. The toxin gene hlyF, was only detected in isolate 8. In all, one VAG was detected in isolate 6 (iutA), two VAGs in isolate 5 (iutA, iucD) and three each in isolate 1 (iutA, iucD, ompT) and isolate 8 (iutA,ompT, hlyF). When isolate 1 was inoculated in a healthy chicken, it exhibited symptoms of colibacillosis with visible lesions. Conclusions: The results confirm the importance of VAGs in the pathogenesis of APEC.