The aim of this study was to determine the time for a 3-log CFU/g outgrowth of Staphylococcus aureus and its toxin production in previously frozen precooked tuna meat (albacore [Thunnus alalunga] prepared as loin, chunk, and flake or skipjack [Katsuwonus pelamis] prepared as chunk and flake) held either at 21 or 27°C. A five-strain cocktail of enterotoxin-producing S. aureus was surface inoculated with ~103 CFU/g onto tuna samples. The experimental time–temperature conditions were designed to mimic common industry holding conditions. After a 3-h incubation at 37°C, inoculated samples were individually vacuum sealed and stored at −20°C for 4 weeks. Following frozen storage, samples were thawed to the target temperature (21 or 27°C) and then incubated aerobically. Growth of S. aureus in tuna was then monitored using Baird Parker agar; simultaneously, aerobic plate counts, enterotoxin production, and sensory profile (color and odor) were determined. The results showed that the time for a 3-log CFU/g increase was >20 h at 21°C and 8 to 12 h at 27°C for albacore, with toxin production observed at 14 to 16 h at 21°C and at 8 h at 27°C. A 3-log CFU/g increase for skipjack occurred at 22 to 24 h at 21°C and at 10 to 14 h at 27°C. The toxin production in skipjack started at 20 to 22 h at 21°C and at 8 to 10 h at 27°C. Toxin production was observed before a 3-log increase was achieved in albacore samples at 21°C. Under all conditions, toxins were detected when the cell density of S. aureus was 6 log CFU/g. Overall, significantly faster S. aureus growth was observed in albacore compared with skipjack (P < 0.05), possibly owing to differences in sample composition (e.g., pH and salt content). The data developed from this study can be used by the tuna industry to model the growth and enterotoxin production of S. aureus and to design manufacturing controls that ensure food safety.