Agricultural workers are exposed to high concentrations of airborne microorganisms and thus have an increased risk for developing respiratory diseases. Respirators, when properly selected and used, can decrease the exposures in agricultural environments. In this study, a new field-compatible method was developed to dynamically measure the protection provided by respirators against dust and microorganisms in agricultural environments. This method includes filter sampling to determine the protection provided by respirators against biological particles (fungal and actinomycete spores, bacteria). The new method was evaluated first in the laboratory under controlled conditions. After that, it was tested in the field during different agricultural operations while farmers were using the new N95 filtering-facepiece respirators: animal feeding (dairy, swine and poultry), grain harvesting and unloading, and routine checking of animals in confinements (swine and poultry). Extensive laboratory evaluation confirmed that the sampling system can detect changes in the protection factors caused by variation in faceseal leaks, human activity, and breathing pattern in both manikin-based and human tests. It was found that the sampling flow was least affected by the inhalation flow when the sampling probe was imbedded on the respirator surface. Leak location, leak size, breathing patterns, and exercises did affect the measurement of the protection factors obtained by a N95 filtering facepiece respirator because of the differences in the in-mask airflow dynamics contributed by the leak, filter material, sampling probe, and inhalation. The field testing showed that the workplace protection factors (WPFs) increased with increasing particle size. The WPF for total culturable fungi was significantly greater than for total culturable bacteria. With respect to microorganisms, the WPFs for Cladosporium and total fungi were significantly correlated with the WPFs for particles of the same sizes. As compared to the WPFs for dust particles, the WPFs for bioaerosols were found more frequently below 10, which is a recommended assigned protection factor (APF) for N95 filtering facepiece respirators. More than 50% of the WPFs for microorganisms (mean aerodynamic diameter < 5 microm) were less than the proposed APF of 10. The results of this study provide useful pilot data to establish guidelines for respiratory protection against airborne dust and microorganisms on agricultural farms. The method is a promising tool for further epidemiological and intervention studies in agricultural and other occupational and non-occupational environments contaminated with airborne dust and bioaerosols.