Bacteria form biofilms by adhering to biotic or abiotic surfaces. This phenomenon causes several problems, including a reduction in the transport of mass and heat, an increase in resistance to antibiotics, and a shortening of the lifetimes of modules in bioindustrial fermentors. To overcome these difficulties, we created a biofilm production-deficient Escherichia coli strain, BD123, by deleting genes involved in curli biosynthesis and assembly, Delta(csgG-csgC); colanic acid biosynthesis and assembly, Delta(wcaL-wza); and type I pilus biosynthesis, Delta(fimB-fimH). E. coli BD123 remained mostly in the form of planktonic cells under the conditions tested and became more sensitive to the antibiotics streptomycin and rifampin than the wild-type E. coli MG1655: the growth of BD123 was inhibited by one-fourth of the concentrations needed to inhibit MG1655. In addition, the transformation efficiency of BD123 was about 20 times higher than that of MG1655, and the production and secretion of recombinant proteins were approximately 16% and approximately 25% greater, respectively, with BD123 than with MG1655. These results indicate that the newly created biofilm production-deficient strain of E. coli displays several key properties that substantially enhance its utility in the biotechnology arena.