A simple two-chamber diffusion method was developed to study the diffusion properties of bacteriophages (phages). The apparent diffusion coefficients (D-app) of Myoviridae phage T4 and filamentous phage fNEL were investigated, and the diffusion of the phages was found to he much slower than the diffusion of three antibiotics, ciprofloxacin, penicillin G. and tetracycline. D-app of T4 and fNEL in water through filter paper were calculated to be 2.8 x 10(-11) m(2)/s and 6.8 x 10(-12) m(2)/s, respectively, and D-app of fNEL through agarose gel membrane, an artificial biofilm, was also calculated to be smaller than that of T4. In addition, D-app of phages through agarose gel was dependent on agarose concentration due to the similar size of phage and agarose gel mesh. We concluded that D-app of phages through an artificial biofilm is dependent on both phage morphology and biofilm density, and suggest the use of this method to study diffusion properties through real biofilms. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1213-1221, 2010