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We investigated the impact of peroxyacetic acid (PAA; 200 ppm) spray on the microbiota and shelf life of commercial, vacuum-packed beef stored at chiller temperatures. Ribeye cuts ( n  = 147) were collected from a local beef plant on the day of production for two consecutive days, with one set collected at the start of work with the PAA spray nozzles turned off (control) and during routine production with the PAA spray nozzles turned on (PAA) each day. Packs were stored at 4, 2, and -1°C for up to 34, 104, and 180 days and sampled at appropriate intervals for sensory assessment, microbial enumeration, and microbial profiling by 16S rRNA gene amplicon analysis. Treatment with PAA did not affect the initial meat pH, the initial numbers of total aerobes, lactic acid bacteria, or Enterobacteriaceae ( P > 0.05) before storage; however, it delayed the onset of spoilage by 7, 21, and 54 days at 4, 2, and -1°C, respectively. Square-root models of the variation of growth rate with temperature indicated lactic acid bacteria grew faster and Enterobacteriaceae grew slower on PAA-treated than on untreated meat. Negative associations between pH and deterioration of meat during storage were observed for PAA-treated meat. During storage, the microbiota were primarily dominated by Carnobacterium and Lactobacillus / Lactococcus on control meat but by Leuconostoc on PAA-treated meat. Serratia , Yersinia , and Clostridium were identified by linear discriminant effect size analysis as biomarkers for control meat; Clostridium was found in high abundance in samples that had the highest spoilage scores. IMPORTANCE The findings of this study show that PAA solutions applied at low concentrations under commercial settings positively modulated the meat microbiota. It did not have bactericidal effects for beef subprimals with very low microbial loads. However, it differentially impacted the members of the microbiota, which resulted in delayed onset of spoilage of vacuum-packed beef subprimal stored at all three temperatures (4, 2, and -1°C). This differential impact could be through one or a combination of the following factors: favoring the growth of lactic acid bacteria, which may in turn exert a competitive exclusion that might be due to production of antimicrobial compounds such as organic acids and bacteriocins; exerting synergistic antimicrobial effects with low temperatures against members of Enterobacteriaceae ; and direct or indirect inhibitory effects against members of the clostridia. These findings not only advance our understanding of the microbial ecology of vacuum-packed meat stored at chiller temperatures but also suggest that bacteriostatic concentrations of antimicrobial interventions can be explored for shelf-life extension., (© Crown copyright 2021.)