Multidrug-resistant Enteroaggregative Escherichia coli (EAEC) is gaining recognition as an important diarrheagenic pathogen with enhanced virulence characteristics. In this study, six typical EAEC isolates from clinical and animal sources were characterized phenotypically and genotypically. All isolates demonstrated the characteristic stacked-brick adherence pattern on HEp-2 cells and were positive for key EAEC-associated genes (aggR, cvd432, fimA, ecp, and irp2). Antimicrobial susceptibility testing identified three isolates as multidrug-resistant (MDR), with resistance observed against antibiotics such as, tetracycline, ampicillin, and ciprofloxacin. ESBL production was confirmed in MDR strains via double-disc synergy, nitrocefin, and PCR assays targeting bla_TEM and bla_CTXM9 genes. Under simulated gut stress conditions (bile salts, acidic pH, and oxidative stress), MDR strains exhibited enhanced survival and faster growth kinetics compared to non-MDR strains. Biofilm assays revealed stronger biofilm formation by MDR strains on diverse surfaces, with confocal microscopy confirming greater bio-volume and viability of MDR biofilms. In vivo survival assays using Galleria mellonella larvae showed significantly higher virulence of MDR strains, with increased mortality rates over 96 hours. The findings highlight the enhanced stress tolerance, biofilm-forming capacity, and pathogenicity of MDR-EAEC strains, underscoring their potential role in persistent infections and public health concerns.