Although fungal endophytes are commonly investigated for their ability to deter microbial plant pathogens, few studies have examined the activity of fungal root endophytes against nematodes. The soybean cyst nematode (SCN, Heterodera glycines Ichinohe), the most severe yield-limiting pathogen of soybean [Glycine max (L.) Merr.], is commonly managed through rotation of soybean with corn (Zea mays L.), a non-host of the SCN. A total of 626 fungal endophytes were isolated from surface-sterilized corn and soybean roots from experimental plots in which soybean and corn had been grown under annual rotation and under 1, 3, 5, and 35 years of continuous monoculture. Fungal isolates were grouped into 401 morphotypes, which were clustered into 108 operational taxonomic units (OTUs) based on 99% sequence similarity of the full ITS region. Morphotype representatives within each OTU were grown in malt extract broth (MEB) and in a secondary metabolite-inducing medium buffered with ammonium tartrate (SMAT), and their culture filtrates were tested for nematicidal activity against SCN juveniles. A majority of OTUs containing isolates with nematicidal culture filtrates were in the order Hypocreales, with Fusarium being the most commonly isolated nematicidal genus from corn and soybean roots. Less commonly isolated taxa from soybean roots included the nematophagous fungi Hirsutella rhossiliensis, Metacordyceps chlamydosporia, and Arthrobotrys iridis. Root endophytic fungal diversity in soybean was positively correlated with SCN density, suggesting that the SCN plays a role in shaping the soybean root endophytic community.