Studies were done at 21°C to determine the bactericidal activity of lactic acid, levulinic acid, and sodium dodecyl sulfate (SDS) applied individually and in combination on Shiga toxin-producing Escherichia coli (STEC) in pure culture and to compare the efficacy of lactic acid and levulinic acid plus SDS treatments applied by spray or immersion to inactivate STEC and Salmonella (10(7) CFU/cm(2)) on beef trim pieces (10 by 10 by 7.5 cm). Application of 3 % lactic acid for 2 min to pure cultures was shown to reduce E. coli O26:H11, O45:H2, O111:H8, O103:H2, O121:H2, O145:NM, and O157:H7 populations by 2.1, 0.4, 0.3, 1.4, 0.3, 2.1, and 1.7 log CFU/ml, respectively. Treatment with 0.5 % levulinic acid plus 0.05 % SDS for <1 min reduced the populations of all STEC strains to undetectable levels (>6 log/ml reduction). Beef surface temperature was found to affect the bactericidal activity of treatment with 3 % levulinic acid plus 2 % SDS (LV-SDS). Treating cold (4°C) beef trim with LV-SDS at 21, 62, or 81°C for 30 s reduced E. coli O157:H7 by 1.0, 1.1, or 1.4 log CFU/cm(2), respectively, whereas treating beef trim at 8°C with LV-SDS at 12°C for 0.1, 1, 3, or 5 min reduced E. coli O157:H7 by 1.4, 2.4, 2.5, or 3.3 log CFU/cm(2), respectively. Spray treatment of beef trim at 4°C with 5 % lactic acid only reduced the E. coli O157:H7 population by 1.3 log CFU/cm(2). Treating beef trim at 8°C with LV-SDS for 1, 2, or 3 min reduced Salmonella Typhimurium by 2.1, 2.6, and >5.0 log CFU/cm(2), respectively. Hand massaging the treated beef trim substantially reduced contamination of both pathogens, with no detectable E. coli O157:H7 or Salmonella Typhimurium (<5 CFU/cm(2)) on beef trim pieces treated with LV-SDS. Reduction of E. coli O157:H7 and Salmonella Typhimurium populations was enhanced, but bactericidal activity was affected by the meat temperature.