Ozone, chlorine and heat applications were compared for killing effectiveness against food spoilage bacteria in synthetic broth. Fresh 24-h bacterial cultures of Pseudomonas fluorescens (ATCC 948), Pseudomonas fragi (ATCC 4973), Pseudomonas putida (ATCC 795), Enterobacter aerogenes (ATCC 35028), Enterobacter cloacae (ATCC 35030) and Bacillus licheniformis (ATCC 14580) were exposed to ozone (0.6 ppm for 1 min and 10 min), chlorine (100 ppm for 2 min) or heat (77 ± 1°C for 5 min). One-minute ozonation had little effect against the bacteria. There were significant differences (P < 0.05) among 10-min ozonation, chlorine or heat inactivation of all bacteria exceptB. licheniformis. Ten-minute ozonation caused the highest bacterial population reduction, with a mean reduction over all species of 7.3 log units followed by heat (5.4 log reduction) and chlorine (3.07 log reduction). Clean, passivated, sterile stainless steel (SS) metal coupons [2.54 × 2.54 cm2, American Society for Testing Materials (ASTM) number 304] were incubated in ultra-high temperature (UHT) sterile milk inoculated with P. fluorescens (ATCC 948), P. fragi (ATCC 4973) and P. putida (ATCC 795) for 24–72 h. After biofilm formation, the SS metal coupons were rinsed with phosphate-buffered saline (1 min) and exposed to ozone (0.6 ppm for 10 min) and chlorine (100 ppm for 2 min). Results indicated that both ozone and chlorine significantly reduced the biofilm bacteria adhered to the SS metal coupons as compared to the control (P < 0.05). However, there was no significant difference (P > 0.05) between ozone and chlorine inactivation of the bacteria with the exception of P. putida. Ozone killed P. putida more effectively than chlorine.