Objectives: To evaluate the cytotoxicity of the mixture of Doc's Best Antibiofilm Cement Powder with Copalite Varnish.  Methods: The fresh mixture, set material and two extracts (in polar and non-polar solvents) prepared using the mixture of Doc's Best Antibiofilm Cement Powder and Copalite Varnish (Cooley & Cooley Ltd., Houston, TX) were evaluated for cytotoxicity using the agar diffusion method.  The sample preparation followed manufacturer's instructions.  Culture media without serum and phenol served as the negative and positive controls, respectively.  L929 fibroblasts (ATCC, Rockville, MD) were cultured at 37°C in a humidified atmosphere of 5% CO₂.  A cell suspension (approximately 1.0x10⁵ cells/mL) was aliquotted into 6-well plates (5 mL/well) and cultured for 24 hours.  The media was withdrawn and an overlay agar was poured over the cell monolayer.  A neutral red solution was placed on the agar surface for approximately 20 minutes and the excess dye was removed.  The samples were placed at the center of agar surface in each well.  Four samples were tested for each group.  Plates were then returned to the incubator.  Cytotoxicity was examined by measuring the zones of cell decolorization and evaluating cell lysis under an inverted microscope after 24 and 48 hours.   Results: The results showed that the positive control, phenol, caused significant cytotoxicity at both 24 and 48 hours and substantial cell lysis while no cell decolorization and lysis were observed in the negative controls.  The fresh mixture and the set mixture of Doc's Best Antibiofilm Cement Powder with Copalite Varnish, as well as the two extracts, did not induce any cell decolorization and lysis.  The observations were comparable to those obtained from the negative controls.  Conclusions: Under conditions of the study, the mixture of Doc's Best Antibiofilm Cement Powder with Copalite Varnish is not cytotoxic as evaluated using the agar diffusion method.