Bioaerosol sampling is necessary to monitor and control human exposure to harmful airborne microorganisms. An important parameter affecting the collection of airborne microorganisms is the electrical charge on the microorganisms. Using a new design of an electrostatic precipitator (ESP) for bioaerosol sampling, the polarity and relative strength of the electrical charges on airborne microorganisms were determined in several laboratory and field environments by measuring the overall physical collection efficiency and the biological collection efficiency at specific precipitation voltages and polarities. First, bacteria, fungal spores, and dust dispersed from soiled carpets were sampled in a walk-in test chamber. Second, a simulant of anthrax-causing Bacillus anthracis spores was dispersed and sampled in the same chamber. Third, bacteria were sampled in a small office while four adults were engaged in lively discussions. Fourth, bacteria and fungal spores released from hay and horse manure were sampled in a horse barn during cleanup operations. Fifth, bacteria in metalworking fluid droplets were sampled in a metalworking simulator. It was found that the new ESP differentiates between positively and negatively charged microorganisms, and that in most of the tested environments the airborne microorganisms had a net negative charge. This adds a signature to the sampled microorganisms that may assist in their identification or differentiation, for example, in an anti-bioterrorism network.