Colibacillosis, caused by avian pathogenic Escherichia coli (APEC), represents a major threat to poultry production, leading to significant mortality and economic losses. This study aimed to characterize an APEC strain, HPVN24, isolated from diarrheic chickens at a farm in Hai Phong, Vietnam. The strain was investigated through phenotypic assays, antibiotic susceptibility profiling, and whole-genome sequencing using the Illumina platform. HPVN24 exhibited β-hemolytic activity and resistance to trimethoprim, ampicillin, and ciprofloxacin. Whole-genome analysis identified the strain as serotype O78:H9 and sequence type ST23, with a genome size of 5.05 Mb and a GC content of 50.57%. Genome annotation revealed a wide repertoire of genes involved in metabolism, secretion systems, virulence, and biofilm formation. Virulence-associated genes included those related to adhesion, iron acquisition, hemolysin production, and stress response. Analysis predicted multidrug resistance to 18 antibiotic classes, with particularly strong resistance to fluoroquinolones. Phylogenetic comparison demonstrated that HPVN24 clustered closely with O78:H9 strains isolated from poultry in other regions, suggesting potential transmission across populations. These findings indicate that HPVN24 is a multidrug-resistant and highly virulent APEC strain linked to colibacillosis outbreaks in Vietnam and highlight the need for ongoing surveillance, judicious antibiotic usage, and alternative strategies to ensure poultry health and food safety.