The ability of Listeria monocytogenes and two competitive exclusion (CE) bacteria, Lactococcus lactis subsp. lactis strain C-1-92 and Enterococcus durans strain 152, to form biofilms on coupons composed of different materials (stainless steel, plastic, rubber, glass, and silicone) was determined at 4 and 8 °C. Biofilm characteristics were determined by scanning electron microscopy. L. monocytogenes produced well-formed biofilms within 24 h at 37 °C on coupon surfaces. Treating Listeria-laden biofilms with the CE isolates individually at either 4 or 8 °C for 3 weeks substantially reduced or eliminated listeriae in the biofilms. Treatment with L. lactis subsp. lactis strain C-1-92 and E. durans strain 152 at 4 °C for 3 weeks reduced the population of L. monocytogenes in a biofilm from 7.1 to 7.7 log CFU/cm2 to 3.0 to 4.5 log CFU/cm2 and to 3.1 to 5.2 log CFU/cm2 , respectively, and treatment at 8 °C for 3 weeks reduced L. monocytogenes from 7.5 to 8.3 log CFU/cm2 to 2.4 to 3.5 log CFU/cm2 and to 3.8 to 5.2 log CFU/cm2, respectively, depending on the coupon composition. These two CE isolates were combined and evaluated for control of Listeria bacteria in floor drains of a ready-to-eat poultry processing plant. The results revealed that treating the floor drains with CE four times in the first week eliminated detectable Listeria bacteria from five of six drains, and the drains remained free of detectable Listeria bacteria for 13 weeks following the first four treatments. These studies indicate that CE can effectively reduce Listeria contamination in biofilms and in flow drains of a plant producing ready-to-eat poultry products.