Introduction: Preventing healthcare professionals from acquiring occupational infectious diseases is very important in maintaining healthcare delivery systems. For protection in the work place, healthcare professionals use PPE which helps prevent exposure to pathogens during patient care. N95 respirators protect healthcare workers against airborne pathogens that are known to be associated with different respiratory diseases. Since previous studies have shown that viruses can survive on PPE surfaces, it is important to examine the survival of viruses on respirators to determine if reuse of the same N95 respirator is possible when PPE shortages occur. Goal: The goal of this research is to determine the inactivation of bacteriophage Φ6 on the surface of N95 respirators at ambient temperature and two different relative humidity levels, 40 and 60%. Result: The linear regression showed that rate of inactivation was much lower in 40% than 60% RH (40%: Slope= -0.046± 0.007040; 60%: Slope= -0.20± 0.006136). Over 24 hours, there was a ~1 Log10 reduction in virus at 20°C and 40% RH, while there was a ~4 Log10 reduction at 20°C and 60% RH. Within the timeframe of a single patient encounter, there was a <0.02 Log10 reduction in virus at 40% RH and a <0.1 Log10 reduction at 60% RH. Conclusion: Bacteriophage Φ6 survives on N95 respirators for up to 24 hours at ambient temperature and 40 and 60% relative humidity levels. Inactivation rate was lower in 40% than 60% RH. The results showed that enveloped viruses survive on the surface of N95 respirators for longer than a single patient encounter. Therefore, this should be taken into consideration when doing a risk assessment of reusing N95 respirators when shortages occur.