The second messenger 3'aEuro"5'-cyclic diguanylic acid (c-di-GMP) promotes biofilm formation, and c-di-GMP is synthesized by diguanylate cyclases (characterized by a GGDEF domain) and degraded by phosphodiesterases. Here, we evaluated the effect of the 12 E. coli GGDEF-only proteins on biofilm formation and motility. Deletions of the genes encoding the GGDEF proteins YeaI, YedQ, YfiN, YeaJ, and YneF increased swimming motility as expected for strains with reduced c-di-GMP. Alanine substitution in the EGEVF motif of YeaI abolished its impact on swimming motility. In addition, extracellular DNA (eDNA) was increased as expected for the deletions of yeaI (tenfold), yedQ (1.8-fold), and yfiN (3.2-fold). As a result of the significantly enhanced motility, but contrary to current models of decreased biofilm formation with decreased diguanylate cyclase activity, early biofilm formation increased dramatically for the deletions of yeaI (30-fold), yedQ (12-fold), and yfiN (18-fold). Our results indicate that YeaI, YedQ, and YfiN are active diguanylate cyclases that reduce motility, eDNA, and early biofilm formation and contrary to the current paradigm, the results indicate that c-di-GMP levels should be reduced, not increased, for initial biofilm formation so c-di-GMP levels must be regulated in a temporal fashion in biofilms.