Arbuscular mycorrhizae (AM) fungi can increase the biomass of host plants that are used as biofuel feedstock. However, little is known about the effects of AM fungi during aqueous ammonia pretreatment of biomass to remove lignin or on the enzymatic hydrolysis of cellulose. The analysis of mycorrhizal colonization (Rhizophagus irregularis or Glomus versiforme) on stems of black locust (Robinia pseudoacacia) plants in their first and second year of growth revealed that the presence of AM fungi and the growth time significantly influenced the lignin and cellulose content of untreated black locust but had no effect on the content of stems pretreated with aqueous ammonia. The presence of AM fungi and/or the growth time also affected the black locust stem structure and the chemical structure of lignin. Hydrolysis of mycorrhizal and non-mycorrhizal biomass produced similar glucose yields (except for the second-year R. irregularis biomass, which produced significantly less glucose than the other treatments). The results suggest that mycorrhizal black locust biomass is a suitable substrate for biofuel production.