Avian pathogenic Escherichia coli (APEC) is one of the pathogens that most concerns the poultry industry worldwide due to the economic losses it can cause. APEC persistence and survival, both in the environment and in the host, may be a consequence of biofilm-producing capabilities. The aim of this study was to evaluate APEC strains' biofilm production and its relationship to in vivo pathogenicity. Two hundred thirty-eight APEC isolates from three different origins (broiler bedding material, cellulite lesions, and respiratory diseases) were selected. The in vivo pathogenicity index (PI) was determined. Biofilm formation was evaluated using a microplate assay with analysis of colony morphology in Congo Red agar in order to detect the phenotypic expression of curli fimbriae and cellulose. Regarding biofilm production, it was observed that 55.8% of the strains produced biofilms. In the morphological test, 88.2% of the isolates expressed one or both components at one of the temperatures at least, and 11.8% of the isolates did not express curli or cellulose. Cellulose production was significantly higher at 25 °C. On the other hand, curli production was significantly higher at 37 °C. The study data indicate that there is no association between biofilm production and in vivo pathogenicity.