The major bifunctional transpeptidases/transglycosylases of Escherichia coli, penicillin-binding proteins (PBPs) 1a and 1b, were evaluated for their influence on biofilm formation. While the PBP1a mutant was unaffected, the PBP1b mutant exhibited significantly decreased biofilm formation and motility. Interestingly, the extracellular indole concentration was higher in the PBP1b mutant, and similar phenotypic defects were replicated in the wild-type upon addition of exogenous indole. Expression of PBP1b in trans substantially decreased indole production and restored normal phenotypes. Results further suggest that rpoS deletion has a counteracting effect on the mrcB mutant. These findings indicate that PBP1b deletion influences biofilm formation and motility, possibly through indole.