Based on current U.S. Food and Drug Administration acidified foods guidelines, regulatory approval of commercial pickled egg production without a final heat treatment requires challenge studies. We conducted challenge studies to verify common pickled egg processing parameters. Hard-boiled eggs were acidified in ambient temperature brine at a 60:40 egg/brine ratio. Four acidification treatments were studied in triplicate: 5% acetic acid (AA) or 2.5% AA brine with and without 0.05% sodium benzoate. These treatments resulted in 2% or 1% AA with or without 0.02% sodium benzoate, respectively, in the total system. Samples were stored at 7 °C until pH at the yolk center was ≤ 4.6; subsequently, samples were held at ambient temperature. Egg pH was measured at 24- to 48-h intervals until equilibrium pH was reached (4.0 and 4.4). Eggs and jar lids were challenged with separate pathogen cocktails (six strains and/or serovars) of Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus. After 5 and 9 days, the pH fell below 4.6 in 2% AA and 1% AA eggs, respectively. Sodium benzoate did not affect acidification rate for these brine treatments (P ≥ 0.05), nor did sodium benzoate affect pathogen die-off. E. coli O157:H7, Salmonella, and L. monocytogenes were undetectable (<1 CFU/g) in pickled eggs in 2% AA at 72 h; S. aureus was undetectable after 7 days. In 1% AA eggs, Salmonella was undetectable after 10 days. No pathogens were detectable after 14 days. No pathogens were detectable on lids within 72 h for the 2% AA treatment. Only S. aureus was detectable on lids after 72 h in the 1% AA treatment and died off rapidly at ambient temperature. Although pathogens began die-off under refrigeration, heat treatment (ambient temperature storage) was required to reach undetectable levels. Minimal inversion was adequate treatment for lids. Pickled eggs should be held under refrigeration for the length of time needed to acidify them to ≤ 4.6 and then held at ambient temperatures to ensure pathogen inactivation.