The hypothesis that ultrasound increases antibiotic transport through biofilms of Escherichia coli and Pseudomonas aeruginosa was investigated using colony biofilms. Biofilms grown on membrane filters were transferred to nutrient agar containing 50 µg/ml gentamicin. A smaller filter was placed on top of the biofilm and a blank concentration disk was situated atop the filter. Diffusion of antibiotic through the biofilms was allowed for 15, 30, or 45 min at 37°C. Some of these biofilms were exposed to 70-kHz ultrasound and others were not. Each concentration disk was then placed on a nutrient agar plate spread with a lawn of E. coli. The resulting zone of inhibition was used to calculate the amount of gentamicin that was transported through the biofilm into the disk. The E. coli and P. aeruginosa biofilms grown for 13 and 24 h were exposed to two different ultrasonic power densities. Ultrasonication significantly increased the transport of gentamicin through the biofilm. Insonation of biofilms of E. coli for 45 min more than doubled the amount of gentamicin compared to their noninsonated counterparts. For P. aeruginosa biofilms, no detectable gentamicin penetrated the biofilm within 45 min without ultrasound; however, when insonated (1.5 W/cm2) for 45 min, the disks collected more than 0.45 µg antibiotic. Ultrasonication significantly increased transport of gentamicin across biofilms that normally blocked or slowed gentamicin transport when not exposed to ultrasound. This enhanced transport may be partially responsible for the increased killing of biofilm bacteria exposed to combinations of antibiotic and ultrasound.