Phanerochaete chrysosporium treatment is less effective as a biological pretreatment on feedstock with larger particle sizes. We hypothesized that the improved effectiveness of the pretreatment when smaller particle sizes are used may be due to the inherently higher bulk density with smaller particle sizes. The effects of substrate bulk density and particle size on the efficacy of P. chrysosporium pretreatment of switchgrass (Panicum virgatum) was tested experimentally. Phanerochaete chrysosporium was grown on senesced switchgrass (2 different particle sizes) with various bulk densities. In all treatments, the fungal-pretreated samples released more glucose during enzymatic saccharification than the control sample. Substrate bulk density was a statistically significant factor in explaining the variation in the amount of glucose released per gram of substrate used. However, the particle size was not found to be a significant factor. On-farm switchgrass pretreatment may not require particle size reduction if the switchgrass is supplied in high-density bales.