Technical Abstract: The continued global increase in the number of cases of Johne’s disease suggests that more information is needed to understand the mechanisms by which the causative agent Mycobacterium avium subsp. paratuberculosis (MAP) is spread among livestock on the farm site. Livestock watering troughs are frequented by all animals on a farm, they provide a moist, nutrient rich environment for bacterial survival and the trough basin provides a surface for bacterial adhesion (i.e., biofilm formation). The goal of this study was to determine if the addition of chlorine to trough water could prevent or reduce biofilms containing MAP on trough materials. MAP was inoculated into trough water containing suspended 3.8 cm x 1.5 cm (1.5 in X 0.6 in) concrete, plastic, galvanized or stainless steel trough materials. Chlorine (2 ppm) was added to the trough water on day 7 and then weekly for 70 days. The concentration of MAP on the trough materials was measured using quantitative, real-time PCR to target the MAP-specific IS900 sequence in DNA extracts. Chlorination was most effective against MAP present on galvanized steel and stainless steel trough materials (99% reduction (t99) in biofilm-associated MAP in 15 and 16 days, respectively). This value was 2 to 4 times higher for cells on plastic and concrete materials (t99 of 33 and 66 days, respectively). Differences in disinfection effectiveness may result from higher pH (pH 8.23) in troughs with concrete materials and lower chlorine availability in troughs with plastic materials. These results suggest that the effectiveness of chlorine disinfection depends on trough material construction, pH and chlorine availability. Optimization of disinfection protocols and elimination of biofilms on trough surfaces should reduce persistence of MAP in trough waters.