To develop a non-thermal method to replace steam autoclaving for white-rot fungi fermentation, Irpex lacteus spawn was inoculated in wheat straw (WSI) or ensiled WS (WSI) at varying ratios of 10%, 20%, 30%, 40%, and 50%, and incubated at 28 °C for 28 days to determine the effects of the ensiling and inoculation ratio on the colonization and degradation ability of Irpex lacteus in wheat straw (WS). The results demonstrate that ensiling effectively inhibited the growth of aerobic bacteria and molds, as well as other harmful microorganisms in WS, which created a favorable condition for the growth of I. lacteus. After the treatment of I. lacteus, the pH of EWSI decreased to below 5, while that of WSI, except for the feedstocks of WSI-50%, was around 7, indicating that I. lacteus colonized well in the ensiled WS because the substrates dominated by I. lacteus are generally acidic. Correspondingly, except for the molds in WSI-50% samples, the counts of other microorganisms in WSI, such as aerobic bacteria and molds, were significantly higher than those in EWSI (p < 0.05), indicating that contaminant microorganisms had a competitive advantage in non-ensiled substrates. Incubation with I. lacteus did not significantly affect the cellulose content of all samples. However, the NDS content of EWSI was significantly higher than that of WSI (p < 0.05), and the hemicellulose and lignin contents were significantly lower than the latter (p < 0.05), except for the NDS and hemicellulose contents of WSI-50% samples. Correlation analysis revealed a stronger negative correlation between NDS content and the contents of hemicellulose, cellulose, and lignin in EWSI, which could be caused by the destruction of lignin and hemicellulose and the conversion from structural carbohydrates to fungal polysaccharides or other compounds in NDS form. Even for WSI-50% samples, the sugar yield of WS treated with I. lacteus improved with an increasing inoculation ratio, but the ratio was not higher than that of the raw material. However, the sugar yield of EWSI increased by 51–80%, primarily owing to the degradation of lignin and hemicellulose. Above all, ensiling improves the colonization ability of I. lacteus in WS, which promotes the degradation of lignin and hemicellulose and the enzymic hydrolysis of cellulose, so combining ensiling and I. lacteus fermentation has promising potential in the pretreatment of WS.