The transmission of some infectious diseases requires that pathogens can survive (i.e., remain infectious) in the environment, outside the host. Relative humidity (RH) is known to affect the survival of some microorganisms in the environment; however, the mechanism underlying the relationship has not been explained, particularly for viruses. We investigated the effects of RH on the viability of bacteria and viruses in both suspended aerosols and stationary droplets using traditional culture-based approaches. Results showed that viability of bacteria generally decreased with decreasing RH. Viruses survived well at RHs lower than 33% and at 100%, whereas their viability was reduced at intermediate RHs. We then explored the evaporation rate of droplets consisting of culture media and the resulting changes in solute concentrations over time; as water evaporates from the droplets, solutes such as sodium chloride in the media become more concentrated. Based on the results, we suggest that inactivation of bacteria is influenced by osmotic pressure resulting from elevated concentrations of salts as droplets evaporate. We propose that the inactivation of viruses is governed by the cumulative dose of solutes or the product of concentration and time, as in disinfection kinetics. These findings emphasize that evaporation kinetics play a role in modulating the survival of microorganisms in droplets.