The environmental sources of microbial aerosols and processes by which they are emitted into the atmosphere are not well characterized. In this study we analyzed microbial cells and biological ice nucleating particles (INPs) in smoke emitted from eight prescribed wildland fires in North Florida. When compared to air sampled prior to ignition, samples of the air–smoke mixtures contained fivefold higher concentrations of microbial cells (6.7 ± 1.3 × 104 cells m−3) and biological INPs (2.4 ± 0.91 × 103 INPs m−3 active at temperatures ≥ −15 °C), and these data significantly positively correlated with PM10. Various bacteria could be cultured from the smoke samples, and the nearest neighbors of many of the isolates are plant epi- and endophytes, suggesting vegetation was a source. Controlled laboratory combustion experiments indicated that smoke emitted from dead vegetation contained significantly higher numbers of cells, INPs, and culturable bacteria relative to the green shrubs tested. Microbial viability of smoke aerosols based on formazan production and epifluorescent microscopy revealed no significant difference in the viable fraction (~80%) when compared to samples of ambient air. From these data, we estimate each fire aerosolized an average of 7 ± 4 × 109 cells and 2 ± 1 × 108 biological INPs per m2 burned and conclude that emissions from wildland fire are sources of viable microbial aerosols to the atmosphere.