Recombinant protein production processes have to maximise yield while minimising cost, which involves balancing plasmid maintenance with cell growth and protein expression. The aim of this study was to analyse the influence of two factors on heterologous protein production in Escherichia coli biofilm cells—the concentration of antibiotic used to maintain the selective pressure and the nutrient medium composition. Escherichia coli JM109(DE3) cells transformed with plasmid pFM23 for enhanced green fluorescent protein (eGFP) expression and containing a kanamycin resistance gene were used. They were exposed to 20 or 30 μg mL−1 kanamycin during biofilm growth in two different culture media, a diluted medium (DM) or the lysogeny broth (LB). The higher antibiotic concentration increased the specific eGFP production in planktonic cells, whereas no increase was detected in biofilm cells. Biofilm formation was increased in DM when compared to LB. Nevertheless, bacteria grown in LB had higher eGFP production than those grown in DM in both planktonic and sessile states (20-fold and 2-fold, respectively). Therefore, among the conditions tested, LB supplemented with 20 μg mL−1 kanamycin was the most advantageous medium to obtain the highest specific eGFP production in biofilm cells.