The human gastric pathogen Helicobacter pylori forms biofilms in vitro and in vivo. The purpose of this study was to evaluate the effects of H. pylori biofilm formation in vitro on clarithromycin (CLR) susceptibility. CLR susceptibility of H. pylori intermediate (2-day) and mature (3-day) biofilms on glass coverslips was determined at concentrations from 0.03 to 0.5 µg/ml. H. pylori biofilm biomass was increased after treatment with CLR at minimum inhibitory concentration levels by up to 4-fold (2-day biofilm) and 16-fold (3-day biofilm). Minimum bactericidal concentrations of CLR against cells in a biofilm were higher (1.0 µg/ml) than that for planktonic cells (0.25 µg/ml). It was shown that the expression of efflux pump genes was significantly increased in biofilm cells. In addition, exposure of biofilms to CLR resulted in high level resistance generation compared to planktonic cells with increased resistance associated with the presence of a point mutation at either position 2142 or 2143 in the domain V loop of the 23S rRNA gene. These results demonstrate that H. pylori biofilm formation decreases the susceptibility to CLR and that H. pylori CLR resistance mutations are more frequently generated in biofilms than in planktonic cells.