Background: Chronically infected wounds represent a major public health problem which is associated with increased morbidity and mortality, particularly with the global threat of antimicrobial resistance. Objectives: Isolation of pathogenic micro-organisms from chronic ulcerative lesions, identification and evaluation of their biofilm formation and antimicrobial resistance pattern. Methodology: This study was carried out on 50 specimens, collected from chronic ulcerative lesions of patients admitted to the Inpatient and Outpatient Surgical Units, Tanta university hospitals. Pathogenic organisms were isolated and identified. Antimicrobial susceptibility was determined, and biofilm formation assay was performed using a tissue culture plate method. Results: The most frequently isolated organisms were Pseudomonas spp. (26.42%), Klebsiella spp. (24.52%) then E. coli (16.98%). All E. coli isolates showed resistance to aztreonam and Ampicillin /sulbactam. Also, all Klebsiella isolates showed resistance to ciprofloxacin, levofloxacin and Ceftazidime. While 92.86% of Pseudomonas isolates showed resistance to Ceftazidime. About 58.5 % of isolated pathogens were biofilm producers with Pseudomonas spp. were the most frequently biofilm producers. Conclusion: The isolated pathogens showed high rate of resistance to most of the tested antimicrobial agents, with high rate of biofilm formation among most of isolates.