The increasing role of carbon nanotubes (CNTs) in various biological applications has led to a number of studies on the cytotoxicity of solution-phase CNTs, but few studies are available concerning the cytotoxicity of CNT films. Herein, we studied the potential health effect of CNT films fabricated with three commercial surfactants (sodium cholate, sodium dodecyl sulfate, and triton X-100). Multi-walled carbon nanotube-surfactant dispersions were coated onto substrates through air-spray technique. Cellular morphology, MTT assays, as well as the expression of TNF-α and IL-1β of RAW 264.7 cells cultured on the spray-coated CNT films were evaluated for cytotoxicity. It was found that the cytotoxicity of the CNT films was largely dependent on the type of surfactant used and could be significantly reduced by mild washing steps. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2011.