Triclosan is a potent biocide that is included in a diverse range of products. This research was aimed to investigate the susceptibility of planktonic and biofilm-associated Salmonella enterica serovar Typhimurium to triclosan, and to identify potential mechanisms of adaptation. The effect of triclosan was studied on planktonic Salmonella (log and stationary phases), on biofilm-associated cells, on bacteria derived from disrupted biofilms and on a biofilm-deficient mutant. An eight-log reduction of exponentially growing cells was observed with 1000 μg mL−1 triclosan within 10 min, a 3.6-log reduction in stationary cells and a 6.3-log reduction in stationary cells of a biofilm-deficient mutant (P<0.05). Biofilm-associated cells were tolerant (1-log reduction). However, biofilm-derived cells showed sensitivity to triclosan similar to stationary-phase cells. Triclosan induced the transcription of fabI and micF. Within biofilms, triclosan also up-regulated the transcription of acrAB, encoding for an efflux pump, marA, and the cellulose-synthesis-coding genes bcsA and bcsE. Thus, Salmonella within biofilms could experience reduced influx, increased efflux and enhanced exopolysaccharides production. Our results demonstrated that the tolerance of Salmonella towards triclosan in the biofilm was attributed to low diffusion through the extracellular matrix, while changes of gene expression might provide further resistance to triclosan and to other antimicrobials.