Limited information is available on how external environmental factors (e.g., the type of carbon source) affect biofilm architecture, conjugative transfer of the plasmid pJP4 and xenobiotic degradation in biofilms. The aim of this project was to investigate the influence of glucose and 2,4-dichlorophenoxyacetic acid, two different carbon sources which represent the absence and presence of selective pressure, respectively, on the combined effect of biofilm architecture, transfer of the plasmid pJP4 in soil derived mixed culture biofilms and consequent biodegradation of 2,4-D. The pJP4 plasmid was transferred to soil-derived mixed culture recipients in plate mating experiments and isolated transconjugant colonies were characterized as Comamonas testosteroni. Donor and transconjugant cells were not detected microscopically in biofilms and no transconjugant colonies were isolated; however, gfp, dsRed, and tfdB genes were detected in biofilm effluents with and without selective pressure. Heterogeneous biofilm architecture was observed for both with and without selective pressure.