Studies were conducted to (i) determine whether inoculants of nonpathogenic Escherichia coli biotype I effectively served as surrogates for E. coli O157:H7, non-O157 Shiga toxin-producing E. coli, and Salmonella when prerigor beef carcass tissue was treated with a commercially available blend of lactic acid and citric acid (LCA) at a range of industry conditions of concentration, temperature, and pressure; (ii) determine the antimicrobial efficacy of LCA; and (iii) investigate the use of surrogates to validate a hot water and LCA sequential treatment as a carcass spray intervention in a commercial beef harvest plant. In an initial laboratory study, beef brisket tissue samples were left uninoculated or were inoculated (-6 log CFU/cm2) on the adipose side with E. coli O157:H7 (5-strain mixture), non-O157 Shiga toxin-producing E. coli (12-strain mixture), Salmonella (6-strain mixture), or nonpathogenic E. coli (5-strain mixture). Samples were left untreated (control) or were treated with LCA, in a spray cabinet, at one of eight combinations of solution concentration (1.9 and 2.5%), solution temperature (43 and 60°C), and application pressure (15 and 30 lb/in2). In a second study, the E. coli surrogates were inoculated (-6 log CFU/cm2) on beef carcasses in a commercial facility to validate the use of a hot water treatment (92.2 to 92.8°C, 13 to 15 lb/in2) followed by an LCA treatment (1.9%, 50 to 51.7°C, 13 to 15 lb/in2, 10 s). In the in vitro study, surrogate and pathogen bacteria did not differ in their response to the tested LCA treatments. Treatment with LCA reduced (P < 0.05) inoculated populations by 0.9 to 1.5 log CFU/cm2, irrespective of inoculum type. The hot water and LCA sequential treatments evaluated in the commercial facility reduced (P < 0.05) the inoculated nonpathogenic E. coli surrogates on carcasses by 3.7 log CFU/cm2. This study therefore provides the meat industry with data for this sequential multiple hurdle system for the operation parameters described.