Biogas slurry with rich nutrients could be applied as fertilizer to improve nitrogen absorption and soil structure. Arbuscular mycorrhizal fungi (AMF) are beneficial soil microorganisms that establish mutualistic relationships with the plant roots. The purpose of this study was to study the effects of AMF and biogas slurry treatment on hybrid Pennisetum growth, soil chemical properties, and soil microorganisms. The results revealed that the biomass yield of hybrid Pennisetum was significantly increased after the application of biogas slurry, and it reached the peak values when the biogas slurry dosage was 900 t/hm², which were 13,216.67 kg/hm² and 13,733.33 kg/hm² in AMF− and AMF+ treatment groups, respectively. Moreover, biogas slurry treatment has a significant promoting effect on other agronomic traits related to biomass yield. As for soil chemical indicators, the contents of total nitrogen, nitrate nitrogen, ammonia nitrogen, and available phosphorus in the soil increased with the increase in biogas slurry application, while the soil organic matter was decreased. The addition of arbuscular mycorrhizal fungi significantly increased the species diversity of soil fungi with no biogas slurry application. Furthermore, when biogas slurry was applied, it had no significant effect on soil microbial diversity and composition, no matter the AMF+ or AMF− treatment. The research results can provide a reference for the long-term utilization of biogas slurry and it also can be used in the actual production of hybrid Pennisetum.