Liquid dairy manure, which is produced in enormous quantities in flush dairy manure management systems, is commonly used as an alternative to chemical fertilizers. It provides nutrient benefits to crops and soils. While dairy waste is a well-accepted and widely used fertilizer, the presence of indicator organisms and human pathogens in manure may lead to pathogen contamination in crops and soils. This study is focused on the examination of ozone gas-based sterilization. In the past, ozone (O3) has been used for sanitizing various foods and solid surfaces, but the potential of O3 for eliminating human pathogens in liquid dairy waste is not studied yet. Pathogens such as Salmonella Typhimurium and Escherichia coli O157:H7 are reported to be present in liquid dairy manure, and this research evaluated the effects of various levels of ozone on the survival of these two pathogens. We designed a continuous type O3 treatment system that has four major components: (1) ozone generator using oxygen; (2) ozone concentration control by mixing with pure air; (3) continuous monitoring of ozone concentrations; and (4) ozone experiment chambers. Various levels of ozone (43.26, 87.40, and 132.46 mg·L−1) were produced in the ozone system, and subsequently, ozone was diffused through liquid manure. Liquid manure was exposed to ozone for multiple durations (30, 60, and 120 min). To determine the effectiveness of O3 in eliminating pathogens, time-series samples were collected and analyzed for determining the levels of S. typhimurium and E. coli O157:H7. Preliminary results showed that ozone concentrations of 132.46 mg/L, and exposure time of 120 min resulted in the reduced levels of E. coli and Salmonella. Low levels of ozone and limited exposure time were found to be less effective in pathogen removal potentially due to high solid contents. Additional studies carrying out experiments to evaluate the impacts of solids in combination with ozone concentrations will provide further insights into developing full-scale ozone-based treatment systems.