Escherichia coli (E. coli) represents a significant etiological agent of colibacillosis in poultry, resulting in considerable economic losses for the global poultry sector. The present study aimed to determine molecular characterization, antibiotic resistance, and biofilm formation of E. coli strains isolated from diseased broilers from four provinces of China. A total of 200 tissue samples were collected from the intestine, liver, crop, heart, and spleen and processed for microbiological examination. Molecular detection of E. coli strains, virulence genes, and serotypes was performed using polymerase chain reaction (PCR). Antibiotic susceptibility testing and biofilm formation were assessed using disk diffusion and 96-well microtiter plate assays. The study retrieved 68% (136/200) of E. coli strains from collected samples. Most of the E. coli strains were resistant to enrofloxacin (56%), followed by cefepime (54%), amoxicillin/clavulanate (52%), streptomycin (50%), ampicillin (48%), clindamycin (47%), kanamycin (41%), polymyxin B (37%), tetracycline (35%), sulfamethoxazole/trimethoprim (33%), ceftazidime (31%), meropenem (4.7%), and florfenicol (2.9%). Similarly, the E. coli strains tested positive for at least one virulence gene and specific serotypes. Among these, O145 was the most prevalent serotype, identified in 22 isolates (16.2%), followed by O8 (12.5%), O102 (11.8%), and O9 (11.0%). The tsh gene (10.2%) was the most prevalent virulence gene. This study found that 47.1% of E. coli strains were biofilm-producing, with 62.5% exhibiting weak biofilm production, 29.7% mild biofilm production, and 7.8% strong biofilm production. Similarly, 24.2% of the E. coli strains were avian pathogenic E. coli strains due to the presence of five or more virulence genes, specifically tsh, ompA, fimC, iss, fyuA, and astA, in a single strain by multiplex PCR. The present study recommends continuous surveillance and effective control measures to reduce the burden of avian pathogenic E. coli-related infections in poultry.