The purpose of this research was to characterize Listeria monocytogenes from several environmental and clinical sources and assess the efficacy of single and combined physico-chemical treatments in reducing biofilm on lettuce leaves. PCR analysis of L. monocytogenes isolates collected from different clinical (10 strains) and environmental sources (12 strains) was used to look for the presence of one Listeria-specific gene and five virulence genes. Biofilms of L. monocytogenes were developed on lettuce leaves over 24 h. A 5-min ultrasound and a 300-ppm sodium hypochlorite (NaOCl) wash resulted in similar reductions in cell numbers of 0.82 log CFU cm-2. For chlorine dioxide (ClO2) at 60 ppm, the cell numbers were reduced by ∼5.45 log CFU cm-2. A combined treatment of 5 min of ultrasound plus 300 ppm NaOCl or 40 ppm ClO2, provided maximal efficacy, reducing the number of L. monocytogenes on the lettuce surface to non-detectable levels. Therefore, ClO2 has the potential to replace NaOCl for the disinfection of food products in the food industry.