Microfluidic devices were used to study the influences of hydrodynamics of local microenvironments on Staphylococcus epidermidis (S. epidermidis) biofilm formation and the effects of a poly(beta-1,6-N-acetyl glucosamine)-hydrolyzing enzyme (dispersin B) and/or an antibiotic (rifampicin) on the detachment of the biofilm. Elongated, monolayered biofilm morphologies were observed at high flow velocity and fluid shear locations whereas large clump-like, multilayered biofilm structures were produced at low flow velocity and fluid shear locations. Upon dispersin B treatment, most of the biofilm was detached from the microchannel surface. However, a trace amount of bacterial cells could not be removed from corner locations most likely due to the insufficient wall shear stress of the fluid at these locations. Dispersin B or rifampicin treatment was effective in delaying the dispersal behavior of bacterial cells, but could not completely remove the biofilm. Combined dynamic delivery of dispersin B and rifampicin was found to be effective for complete removal of the S. epidermidis biofilm.