Pediococcus pentosaceus is a lactic-acid producing bacterium inoculated in hay to prevent hay deterioration. This study sought to verify the effect of this treatment on the barn microenvironment. Air samples were obtained from 19 barns using bacterial hay treatment and from 18 control barns with six-stage Andersen samplers and all-glass impingers. Appropriate culture media were used for the recovery and identification of microorganisms. Endotoxins were measured with chromogenic Limulus amoebocyte lysate assay. Median values (respectively for treated and untreated hay barns) were: 5.28 × 105 and 3.84 × 105 colony-forming units (CFU)/m3 for total bacteria; 3.18 × 106 and 4.5 × 106 CFU/m3 for molds; 1.36 × 103 and 1.74 × 103 endotoxin units/m3 for endotoxin levels; and 1.03 × 103 and 3.00 × 103 CFU/m3 for Saccharopolyspora rectivirgula. No viable P. pentosaceus were recovered. The presence of S. rectivirgula, the causative agent for farmer's lung, was not influenced by the hay treatment. Since no significant difference was observed in any of the airborne contaminants, this type of hay treatment probably does not protect farmers from the respiratory effect of ambient microbial contaminants.