Avian colibacillosis, caused by avian pathogenic Escherichia coli (APEC), represents a major threat to the poultry industry, leading to significant economic losses. This study aimed to characterize selected biofilm-producing APEC strains isolated from diseased chickens in the Tizi-Ouzou region of Algeria and to explore potential associations between antimicrobial resistance and the presence of virulence factors. Twenty-four confirmed biofilm-producing E. coli isolates were analyzed for serotype distribution, antimicrobial resistance patterns and virulence gene profiles. While none belonged to the O157 serogroup, all isolates demonstrated concerning resistance profiles, with high rates observed for tetracycline (83.3%), ampicillin (75%), and ciprofloxacin (62.5%). Notably, 40% of the strains exhibited biofilm-forming capability, predominantly showing weak to moderate production levels.Virulence gene profiling revealed traT, bcsA, and csgA as nearly ubiquitous (95.8%), with fimH present in 83.3% of isolates. Intermediate prevalence was noted for iutA (62.5%), fliC (45.8%), and agn43 (33.3%), while fyuA (29.2%) and several other virulence markers (kpsMT II, papC, cnf1, ibeA) occurred at lower frequencies (< 10%). Statistical analysis identified significant correlations between virulence gene content and phenotypic characteristics, including a positive association between virulence gene number and biofilm intensity (p < 0.05). Moreover, the fimH gene showed a strong positive correlation with resistance to the antibiotic nalidixic acid. Resistance to β-lactam antibiotics (cefotaxime, cefepime, aztreonam) was positively correlated (p < 0.05) with papC and ibeA, but negatively correlated with csgA. These findings underscore the complex interplay between antimicrobial resistance and virulence in Algerian biofilm-producing APEC strains, highlighting the need for enhanced surveillance programs and tailored intervention strategies. This study provides critical baseline data for developing effective control measures against colibacillosis poultry production systems.