The transient diffusion of fluorescent tracers into biofilm cell clusters of Staphylococcus epidermidis was visualized by time lapse confocal scanning laser microscopy. Rhodamine B diffused into the center of cell clusters that were 200 to 600 microm in diameter within a few minutes. The apparent effective diffusion coefficient calculated from these data averaged 3.7 x 10(-7) cm(2) s(-1) or 11% of the value in pure water. Fluorescein diffused into biofilm more rapidly, with a diffusion coefficient that averaged 1.6 x 10(-6) cm(2) s(-1), or 32% of the value in water. This study provides direct, visual confirmation that solutes the size of many antibiotics and biocides can diffuse rapidly into biofilms.