Contamination of surfaces in direct contact with food in the processing line is the major cause of finished product cross-contamination, especially conveyor belt surfaces that transfer food products. Polyesterurethane (PSU) is one of the materials widely used in conveyor belt systems; therefore, the objective of this study was to examine the effect of PSU conveyor belt surface roughness on the ability of biofilm formation by two strains of Listeria monocytogenes, i.e. ATCC19114 and ATCC51782. Additionally, the efficiency of a cleaning and sanitizing procedure adopted by a chicken meat plant in Thailand in eliminating biofilm formation on belt surfaces was assessed. The roughness values (Ra) were 0.05 ± 0.00 μm and 1.44 ± 0.01 μm for new (unused) and old conveyor belts (in use for 5 years), respectively. It was found that at 30 °C, both strains of L. monocytogenes formed robust biofilms regardless of differences in surface roughness. Conversely, at 15 °C, the ability of L. monocytogenes to form biofilms decreased with the lower Ra value. Under a simulated processing environment, cleaning and sanitizing reduced L. monocytogenes biofilms from both old and new conveyor belt surfaces to certain levels; however, a total reduction could not be achieved. In addition, more highly concentrated cleaning and sanitizing agents resulted in greater reduction. Results from this study clearly illustrate that surface roughness of material is also an important factor which may hinder effective cleaning and sanitizing. Therefore, the roughness value may be used as an indicator for evaluating the life of conveyor belts being used in the food industry.