In this descriptive study, we used Escherichia coli twin-arginine translocase (tat) mutants to distinguish antibiotic tolerance from the formation of mature biofilm structure. Biofilm formation by wild-type and deltatat strains of E. coli was evaluated using viable cell counts, scanning electron microscopy, and confocal laser-scanning microscopy. Escherichia coli deltatat mutants had an impaired ability to form biofilms when grown in rich or minimal media. These mutants produced disorganized layers and cell aggregates with significantly decreased cell density relative to the wild-type strain. In contrast, wild-type E. coli grown under similar test conditions formed highly structured, surface-adherent communities. We thus determined if this decreased biofilm formation by E. coli deltatat mutants may result in lowered tolerance to antimicrobials. When grown in rich media, planktonic deltatat mutants were hypersensitive to some metals, detergents, and antibiotics. However, the corresponding biofilms were about as resilient as the wild-type strain. In contrast, both planktonic cells and biofilms of the deltatatABC strain grown in minimal media were hypersensitive to many antimicrobials. Remarkably, these biofilms remained up to 365 times more tolerant to beta-lactams than corresponding planktonic cells. Our data suggest that the twin-arginine translocase may play a contributing role in the antimicrobial tolerance, structural organization, and formation of mature E. coli biofilms under nutrient-limited conditions. However, the high tolerance of the deltatatABC strain to bactericidal concentrations of antimicrobials indicates that mature biofilm structure may not be required for surface-adherent E. coli to survive exposure to these lethal factors.