Escherichia coli is a major player in foodborne illnesses, capable of forming biofilms on dairy facilities, leading to milk contamination. Thus, we examined the capacity of E. coli strains from raw milk bulk tanks to form biofilms on surfaces made of polystyrene, stainless steel, and silicone; the potential links between biofilm formation with genes responsible for fimbriae and virulence factors of extra-intestinal E. coli (ExPEC); and the susceptibility of biofilm-forming isolates to iodine and chlorhexidine digluconate. Out of 149 E. coli strains, 42.28% (63/149) formed biofilm on polystyrene, 56.38% (84/149) on silicone, and 21.48% (32/149) on stainless steel. The frequency of genes was: fimH (100%), hlyA (5.4%), irp2 (2.7%), sitA (10.7%), ompT (43.6%), and traT (98%). No biofilm developed when disinfectants were used prior to biofilm formation. However, iodine and chlorhexidine digluconate allowed 25.40% (16/63) of isolates displaying growth after a mature biofilm was formed. The presence of biofilm on different surfaces emphasizes the vital need for thorough equipment cleaning, both in farms and in industrial dairy settings. Rapid disinfection is crucial, given the reduced susceptibility of potentially pathogenic E. coli after biofilm maturity.