Biofilms of the homoserine-lactone-defective strain Chromobacterium violaceum CV026 were grown on silicone surfaces in a flow chamber. The effect of medium flow rate, different levels of N-butanoyl-homoserine lactone, and nutrients on biofilm activity was studied by quantifying the proteins and exopolysaccharides attached to the cells. To compare the effect of each of the three variables within a wide range, the experiments were designed as a full factorial search with three levels for each variable. Calculated contour plots demonstrated that N-acyl homoserine lactone is an important determinant of both the amount and the composition of the compounds excreted into the medium.