A high reactivity and leaving no harmful residues make ozone an effective disinfectant for farm hygiene and biosecurity. Our objectives were therefore to (1) characterize the killing capacity of aqueous and gaseous ozone at different operational conditions on dairy cattle manure-based pathogens (MBP) contaminated different surfaces (plastic, metal, nylon, rubber, and wood); (2) determine the effect of microbial load on the killing capacity of aqueous ozone. In a crossover design, 14 strips of each material were randomly assigned into 3 groups, treatment (n = 6), positive-control (n = 6), and negative-control (n = 2). The strips were soaked in dairy cattle manure with an inoculum level of 107-108 for 60 minutes. The treatment strips were exposed to aqueous ozone of 2, 4, and 9 ppm and gaseous ozone of 1and 9 ppm for 2, 4, and 8 minutes exposure. 3M™ Petrifilm™ rapid aerobic count plate and plate reader were used for bacterial culture. On smooth surfaces, plastic and metal, aqueous ozone at 4 ppm reduced MBP to a safe level (≥5-log10) within 2 minutes (6.1 and 5.1-log10, respectively). However, gaseous ozone at 9 ppm for 4 minutes inactivated 3.3-log10 of MBP. Aqueous ozone of 9 ppm is sufficient to reduce MBP to a safe level, 6.0 and 5.4- log10, on nylon and rubber surfaces within 2 and 8 minutes, respectively. On complex surfaces, wood, both aqueous and gaseous ozone at up to 9 ppm were unable to reduce MBP to a safe level (3.6 and 0.8-log10, respectively). The bacterial load was a strong predictor for reduction in MBP (P<0.0001, R2 = 0.72). We conclude that aqueous ozone of 4 and 9 ppm for 2 minutes may provide an efficient method to reduce MBP to a safe level on smooth and moderately rough surfaces, respectively. However, ozone alone may not an adequate means of controlling MBP on complex surfaces.