Pretreatment with white rot fungi is a promising method to enhance the digestibility of lignocelluloses; however, sterilization of feedstocks prior to inoculation is one of the costliest steps. To improve the colonizing ability of white rot fungi under non-sterile condition, Irpex lacteus, Pleurotus ostreatus, and Phanerochaete chrysosporium were inoculated in the wheat straw ensiled for 28 days and incubated for 56 days to determine the changes in microbe counts, organic acid content, chemical composition, and rumen and enzymatic digestibility. Results showed that ensiling produced abundant organic acids and suppressed most microbes in wheat straw. Significant growth of I. lacteus was observed after 3 days of incubation, and molds were only detectable at day 7 in the group. At the end of incubation, aerobic bacteria and lactic acid bacteria decreased by 18% and 38% in the wheat straw treated with I. lacteus, but molds, aerobic bacteria, and lactic acid bacteria thrived in those treated with P. ostreatus and P. chrysosporium. Even more, P. ostreatus and P. chrysosporium increased the lignin content of the ensiled wheat straw by 34% and 65%. However, I. lacteus selectively degraded lignin by 28% and improved the rumen and enzymatic digestibility by 18% and 34%. The finding indicates that ensiling prior to fermentation with I. lacteus is an effective method to control spoilage microbes and to enhance the rumen and enzymatic digestibility of wheat straw.