Borrelia burgdorferi is the etiological agent of Lyme disease. Not much is known about the susceptibility of this organism to chemical disinfection. Current antimicrobial susceptibility test methods, such as those published by the American Society for Testing and Materials (ASTM), usually require assessment of the number of colony forming units (cfu) of growing organisms on plates following exposure to an agent. For fast-growing organisms, plates are ready for counting 1-2 days post plating, while several weeks may be needed for slower growing organisms. Spirochetes, like B. burgdorferi are difficult to grow on solid media and typically require long incubation periods, sometimes up to several weeks, to generate visible colonies. These issues make B. burgdorferi cfu assessment by plate counting difficult and unreliable. Furthermore, Borrelia have a demonstrated capacity for pleomorphic forms, and can exist in spirochete, round body, or biofilm forms, depending on culture conditions. Plate counts, by nature, do not allow for assessment of morphological form changes. Additionally, the susceptibility of B. burgdorferi pleomorphic forms to chemical disinfectants has not been tested. In this study, we used the SYBR GREEN I/Propidium Iodide (SG I/PI) viability assay to rapidly estimate the percent kill of B. burgdorferi pleomorphic forms to chemical disinfection. Planktonic spirochete populations in 30-second treated samples showed viability percent values of: >95% for Hanks balanced salt solution (HBSS), ~60% for distilled deionized H2O (dd H2O), <5% for ACS 200, and 1% for 1% glutaraldehyde (GTA). Solutions containing 70% ethanol (ETH) and 1% hypochlorite (HC) showed no viable spirochetes following treatment. The percent of live round body cells following different treatments were: >99% for HBSS and <25% for dd H2O. ACS 200, 1% GTA, and 70% ETH treatments resulted in <1% live round body forms, whereas HC showed no live round cell forms. The susceptibility of B. burgdorferi biofilms to various treatments was also assayed using a SG I/PI viability stain after 30-minute contact times. The percent of viable organisms (green) in the treated biofilms was estimated by microscopic observations. HBSS controls showed >98% of bacteria in the biofilm were alive, while treated biofilms showed the following percent viabilities: ACS 200 - ~2%, 1% HC - <1%, 5% HC - <1%, 1% GTA - ~10%, 70% ETH - ~ 2%, and dd H2O ~40%. These techniques merged standardized assessment of antimicrobial activity in liquid culture using an ASTM-type kill-time procedure with viability techniques used in antibiotic susceptibility testing to rapidly evaluate the percent kill of B. burgdorferi pleomorphic forms in vitro following disinfectant exposure. These results showed that B. burgdorferi biofilm forms are orders of magnitude more resistant to chemical disinfection than other morphological forms of this organism.