Because nicotine upregulates the growth of most oral bacteria, this in vitro study investigated the antimicrobial effect of chlorhexidinedoped thin film on commercially pure titanium against Fusobacterium nucleatum (F. nucleatum) biofilm supplemented with different concentrations of nicotine (0, 1, and 2 mg/mL). Biofilms were formed on a chlorhexidine-doped thin film on commercially-puretitanium discs and compared to the control groups. Biofilm viability, total biofilm growth using a spectrophotometer, extracellular polysaccharide content, and pH variations were assessed as dependent variables. Data were submitted to ANOVA and Tukey honest significant difference tests (α=0.05). F. nucleatum biofilm growth was inhibited when exposed to chlorhexidine-doped thin film (p<0.05). Biofilm supplemented with nicotine did not impact the synthesis of EPS on the same type of treatment (p>0.05). The pH values were significantly increased with the increase of nicotine concentration (p<0.05). Chlorhexidine-doped thin film was effective in reducing F. nucleatum biofilm supplemented with nicotine.