Biofilms on indwelling medical devices such as central venous catheters result in significant morbidity and mortality and have a substantial impact on healthcare delivery. Because routine systemic treatment of patients with catheter-associated bloodstream infections is often ineffective, due to the tolerance of biofilm organisms on these devices, other strategies such as the antimicrobial lock treatment (ALT) have been used. This approach involves the instillation of high concentrations of the antimicrobial agent directly into the biofilm-containing catheter for exposure (i.e., dwell) times sufficient to eradicate the biofilm. Results from human studies, animal studies, and laboratory studies using in vitro model systems have suggested that eradication of a biofilm is possible, depending on the organisms in the biofilm, biofilm age, the antimicrobial agent used, and the dwell/ duration of the treatment. The most effective antimicrobial agents are those (1) that are less affected by the extracellular polymeric substance matrix of the biofilm, (2) that have a more rapid bactericidal effect, or (3) for which the mechanism of action is not dependent upon the growth rates of the organisms. Combining agents may also provide synergy. Fungal biofilms have proven to be much more difficult to treat using the ALT, though newer fungicidal drugs such as the echinocandins hold promise in this regard. However, a serious drawback with the ALT is the potential for the development of resistance. Newer treatments, incorporating agents not classified as antibiotics, appear to effectively eradicate biofilms in in vitro models and should be evaluated in animal and patient studies. Promising technologies that incorporate novel approaches such as ultrasound, bacteriophage, quorum-sensing inhibitors, or enzymes may also provide useful approaches in the future.