This paper is to study the effect of cotton fabric’s moisture on steam sterilization process. An experiment is designed to measure the thermal equilibrium time under different moisture concentrations. The results show that a higher moisture concentration leads to a longer thermal equilibrium time, and it does not affect the wet pack’s penetration efficiency when the moisture concentration is below the threshold value of 30%. The paper concludes that a higher humidity is unfavorable for the thermal conduction during the sterilization process.