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Bio-fertilizer application has been proposed as a strategy for enhancing soil fertility, regulating soil microflora composition, and improving crop yields, and it has been widely applied in the agricultural yields. However, the application of bio-fertilizer in grassland has been poorly studied. We conducted in situ and pot experiments to investigate the practical effects of different fertilization regimes on Leymus chinensis growth, with a focus on the potential microecological mechanisms underlying the responses of soil microbial composition. L. chinensis biomass was significantly (P < 0.05) increased by treatment with 6000 kg ha−1 of Trichoderma bio-fertilizer compared with other treatments. We found a positive (R2 = 0.6274, P < 0.001) correlation between bacterial alpha diversity and L. chinensis biomass. Hierarchical cluster analysis and nonmetric multidimensional scaling (NMDS) revealed that soil bacterial and fungal community compositions were all separated according to the fertilization regime used. The relative abundance of the most beneficial genera in bio-fertilizer (BOF) (6000 kg ha−1Trichoderma bio-fertilizer) was significantly higher than in organic fertilizer (OF) (6000 kg ha−1 organic fertilizer) or in CK (non-amend fertilizer), there the potential pathogenic genera were reduced. There were significant negative (P < 0.05) correlations between L. chinensis biomass and the relative abundance of several potential pathogenic genera. However, the relative abundance of most beneficial genera were significantly (P < 0.05) positively correlated with L. chinensis biomass. Soil properties had different effects on these beneficial and on these pathogenic genera, further influencing L. chinensis biomass. [ABSTRACT FROM AUTHOR]