Aerosolization of bacteriophage MS2 virions by nebulization and charge-reduced electrospray were compared during testing of three filter media. Sample swatches were taken from a surgical mask, N95 filtering-facepiece respirator (FFR), and N100 FFR for use in repeated, short-duration (15 min) penetration tests with bacteriophage MS2 aerosolized by nebulizer and electrospray. Evaluated were (1) the virus suspension preparation protocol, (2) resulting particle size distribution, count stability, and count variability, and (3) the ability to generate culturable MS2 virions. While preparation of the electrospray bacteriophage suspension required additional purification and concentration steps and took more time than the nebulization protocol, it resulted in a much higher titer suspension. The nebulizer produced a polydisperse aerosol; conversely, the electrospray produced a relatively monodisperse aerosol with a count peak at the mobility size of the single virion. The nebulized aerosol particle count was 2.8 times as variable as the electrosprayed aerosol particle count although neither aerosolization method maintained a constant count over repeated 15-minute filter tests. No differences in filter penetration were observed between nebulized and electrosprayed MS2 aerosol particles. Electrosprayed dextrose particles, used as an inert aerosol particle comparator, exhibited higher penetration than MS2 particles in two of the three filter samples, which can be attributed, at least partially, to the difference in dielectric properties of dextrose and virus particles. Both aerosolization methods generated culturable MS2 virions with the electrospray producing an airborne concentration approximately 20-fold higher than the nebulizer. In general, the electrospray produced cleaner, more stable, and more viable bacteriophage aerosol particles compared to conventional nebulization. The findings of this study are expected to assist researchers in selecting appropriate generation methods when using viable virus-based challenge aerosol particles.