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Antibiotic resistance in Escherichia coli has emerged as a serious and growing threat to public and environmental health and domestic wells have been fingered as a potential reservoir of resistant E. coli due to faecal contamination, and are ideal for microbial biofilm formation. Therefore, this study was carried out to investigate biofilm production among 60 multidrug-resistant (MDR) E. coli recovered from domestic wells in Ile-Ife, Nigeria. All biofilm positive isolates were screened for seven associated (papC, iroN, biofilm, fimH, cna, fbna and bcsa) genes by multiplex polymerase chain reaction (PCR). The diversity of the isolates was investigated using (GTG)5-PCR, and a dendrogram was drawn with interactive-tree-of-life software. Overall, 45 of 60 (75%) isolates produced biofilms, comprising strong (n=15; 25%), moderate (n=16; 26.7%), and weak (n=14; 23.3%) producers. Moreover, 35 (77.8%) isolates had at least one biofilm gene, 30 (66.7%) had multiple genes, and 5 (11.1%) had one. The biofilm gene (biofilm, fimH, bcsa, iroN) combination was identified in 10 (28.7%) of the 60 isolates. The isolates (n=14; 40%) with moderate biofilm production had the highest number of genes dominated by the biofilm gene (biofilm, fimH, bcsa) combination, while isolates (n=8; 22.9%) with weak production had the least number of genes and comprised mainly the fimH and cna gene combination. The biofilm formers showed 32 distinct fingerprints and were grouped into nine clades, with clade 6 having the most strains (31.6%). The occurrence of MDR biofilm-producing organisms in domestic wells constitutes a public health concern. There is a need to develop strategies to curb faecal contamination of wells to prevent outbreaks of untreatable diseases.