A new system was used to determine the workplace protection factors (WPF) for dust and bioaerosols in agricultural environments. The field study was performed with a subject wearing an N95 filtering facepiece respirator while performing animal feeding, grain harvesting and unloading, and routine investigation of facilities. As expected, the geometric means (GM) of the WPFs increased with increasing particle size ranging from 21 for 0.7–1 μm particles to 270 for 5–10 μm particles (p < 0.001). The WPF for total culturable fungi (GM = 35) was significantly greater than for total culturable bacteria (GM = 9) (p = 0.01). Among the different microorganism groups, the WPFs of Cladosporium, culturable fungi, and total fungi were significantly correlated with the WPFs of particles of the same sizes. As compared with 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 μm) were less than the proposed APF of 10. Even lower WPFs were calculated after correcting for dead space and lung deposition. Thus, the APF of 10 for N95 filtering facepiece respirators seems inadequate against microorganisms (mean aerodynamic size < 5 μm). These results 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 similar occupational and nonoccupational environments.