Temperature and time are two critical parameters in sous vide cooking which directly affect eating quality characteristics and food safety. This study aimed to evaluate physicochemical and microbiological properties of sous vide chicken breast fillets cooked at twelve different combinations of temperature (60, 70, and 80 °C) and time (60, 90, 120, and 150 min). The results showed that cooking temperature played a major role in the moisture content, cooking loss, pH, a* color value, shear force, and thiobarbituric acid reactive substances (TBARS). Increasing cooking temperature caused an increase in cooking loss, lipid oxidation, TBARS, and pH, while moisture content was reduced (p < 0.05). Cooking time played a minor role and only moisture content, cooking loss, and a* color value were affected by this parameter (p < 0.05). Total mesophilic aerobic bacteria, Psychrotrophic bacteria, and Enterobacteriaceae were not detected during 21 days of storage at 4 °C. Cooking at 60 °C for 60 min showed the optimum combination of temperature and time for sous vide cooked chicken breast fillets. The result of this study could be interesting for catering, restaurants, ready-to-eat industries, and homes to select the optimum combination of temperature and time for improving the eating quality characteristics and ensuring microbiological safety.