Introduction: Staphylococcus aureus is normal flora that lives in the skin and Escherichia coli is lives in large intestine. This bacteria has an ability to cause infection disease if the host is not in a good condition. Therefore, research on viability of normal flora was conducted. The objective of this research was to determine the effect of temperature on viability of normal flora bacteria. Methods: The research used pre-experimental design, conducted in 2017 in Balai Laboratorium Kesehatan Pengujian dan Kalibrasi NTB. The sample used in the research is the pure isolate of Staphylococcus aureus and Escherichia coli. Treatment in this research was the temperature difference and its effect. The samples were treated in temperature 500oC, 600oC, 700oC and 800oC for 15 minutes. Results: Showed that the growth of normal flora bacteria still occur in 500oC after treatment. Conclusion: It can be concluded that there is effect of temperature on viability.