Abstract: Biofilm formation in food processing is a public health concern globally. Biofilm formation in a 5 day period by various strains of Listeria on glass and cement was assayed using Crystal Violet Binding Assay (CVBA) and Bacteria Cell Enumeration (BCE) methods. Biofilm assessment was done in five different culture media and at two incubation temperatures (11 and 28±2°C). Scanning Electron Microscopy (SEM) was used to confirm the formation of the biofilms. Listeria organisms developed biofilms on food contact surfaces with greater levels significantly (p<0.05) formed on cement than glass. Significantly (p<0.05) higher biofilm formation occurred at ambient (28±2°C) than refrigeration (11°C) temperature. Biofilm production increased with additional glucose and 2% blood concentrations in the culture media. SEM confirmed the higher levels of biofilm produced on cement than glass. This study showed Listeria biofilms adhere easily to food contact surfaces which is influenced by nutrient and temperature. Regular evaluation of operation cleanliness in local food processing environments is highly necessary.