In this study we used 2 experimental approaches to evaluate the stability of antimicrobial resistance (AMR) phenotypes, selected AMR genes, and selected virulence genes in Enterococcus faecalis during manure storage on a commercial swine farm. Isolates of E. faecalis were obtained directly from fresh fecal material (n = 120) and from the manure storage facility (n = 85) and compared. Tetracycline resistance and the virulence genes cob, esp, eep, and ccf were detected at lower frequency in manure isolates than in fecal isolates. A second approach consisted of immersing in diffusion chambers pure cultures of E. faecalis that varied in their AMR phenotypes and virulence genotypes in the swine manure storage facility for 8 weeks, sampling periodically, and evaluating the recovered strains for changes in their genotypic or phenotypic characteristics. Enterococcus faecalis populations declined exponentially, with rate constants ranging from 0.011 to 0.022 h(-1). Among the AMR and virulence genes examined, 1 AMR gene (sat4) and 7 virulence genes (agrB(fs), cob, cpd, cylB, efaA(fs), enlA, and esp) were lost at low frequencies in the recovered strains. The AMR phenotypes were stable during the incubation, with minimal loss (P > 0.05) of the streptomycin-resistance phenotype. Overall, these results suggest that some attributes of public health significance in populations of E. faecalis decrease in frequency during manure storage.