Aerosol droplet- and airborne-transmitted diseases are an important healthcare concern. The anthrax attacks of 2001, severe acute respiratory syndrome outbreaks in 2003 which resulted in transmission to numerous healthcare personnel (HCP) and concerns about smallpox as a bioterrorist agent have contributed to heightened concern about airborne infectious agents. Respirators and surgical masks can provide respiratory protection against such airborne diseases but their efficacy needs to be assessed. This study describes a method for quantitatively assessing the relative efficiency of respiratory protective equipment (RPE) when challenged with a bioaerosol. Five surgical masks, three N95 respirators and three surgical N95 respirators were evaluated. All are commercially available and used in US healthcare settings. Bacterial aerosols of vegetative Bacillus anthracis strain Sterne 34F2 (a surrogate for pathogenic B. anthracis) were generated with a six-jet Collison nebuliser. To mimic human respiratory breathing, an automated breathing simulator (ABS) calibrated to normal tidal volume and active breathing rate (500 mL/breath and 20 breath/min, respectively) was used. Respirators were placed on manikin head-forms designed for use in cardiopulmonary resuscitation training and used in our investigation as surrogates for HCP. The method showed that a Collison nebuliser could generate monodisperse bacterial aerosol to effectively test RPE total inward leakage. Also, the AGI-30 air samplers, combined with the ABS, provided an accurate method of quantifying RPE relative efficiency. For the 11 RPE this ranged from 34% to 69% with statistically significant differences between several RPE models. We conclude that neither RPE type nor brand name was an indicator of RPE relative efficiency.