The survival and growth of Listeria monocytogenes and spoilage microflora during storage of fresh beef subjected to different decontamination treatments was studied. Fresh beef inoculated with a five-strain mixture of L. monocytogenes (5.18 log CFU/cm2) was left untreated (control) or was immersed (30 s) in hot water (HW; 75°C), 2% lactic acid (LA; 55°C), hot water followed by lactic acid (HW-LA), or lactic acid followed by hot water (LA-HW) and then stored aerobically at 4, 10, and 25°C for 25, 17, and 5 days, respectively. Initial populations of L. monocytogenes were reduced by 0.82 (HW), 1.43 (LA), 2.73 (HW-LA), and 2.68 (LA-HW) log CFU/cm2. During storage, the pathogen grew at higher rates in HW than in control samples at all storage temperatures. Acid decontamination treatments (LA, HW-LA, and LA-HW) resulted in a weaker inhibition of L. monocytogenes (P < 0.05) at 25°C than at 4 and 10°C. In general, the order of effectiveness of treatments was HW-LA > LA > LA-HW > HW > control at all storage temperatures tested. In untreated samples, the spoilage microflora was dominated by pseudomonads, while lactic acid bacteria, Enterobacteriaceae, and yeasts remained at lower concentrations during storage. Brochothrix thermosphacta was detected periodically in only a limited number of samples. Although decontamination with HW did not affect the above spoilage microbial profile, acid treatments shifted the predominant microflora in the direction of yeasts and gram-positive bacteria (lactic acid bacteria). Overall, the results of the present study indicate that decontamination with LA and combinations of LA and HW could limit growth of L. monocytogenes and inhibit pseudomonads, which are the main spoilage bacteria of fresh beef stored under aerobic conditions. However, to optimize the efficacy of such treatments, they must be applied in the appropriate sequence and followed by effective temperature control.