A number of fungi have been shown to have negative effects on plant-parasitic nematodes. Most of these fungi have been isolated from soil, plant roots, or nematodes themselves. Fungi associated with crops can provide a diverse pool of candidates to test for antagonistic effects against plant parasites and other stressors. We used a hierarchical two-tiered approach to evaluate the efficacy and repeatability of 55 strains of fungi originally isolated as foliar facultative endophytes from upland cotton (Gossypium hirsutum) along with one commercial isolate of Beauveria bassiana for in planta antagonistic effects on root-knot nematodes (Meloidogyne incognita). All fungi were inoculated to cotton using a seed treatment. The number of root galls was quantified 3 weeks after egg inoculation of cotton seedlings. The majority of the fungi tested reduced the number of root galls relative to those on untreated control plants. To assess repeatability, 22 strains that exhibited the strongest reductions in gall numbers were further tested in replicate follow-up assays. Ninety-five percent (21/22) of these retested strains significantly reduced galling in the follow-up assay. Strains that reduced galling the most belonged to the genera Alternaria, Chaetomium, Cladosporium, Diaporthe, Epicoccum, Gibellulopsis, and Purpureocillium. On the contrary, three strains in the genera Alternaria and Curvularia significantly increased gall numbers. Our results indicate that a large proportion of the fungal strains originally isolated from cotton as naturally occurring foliar facultative endophytes are capable of reducing root-knot nematode infection when applied back to the plant as a seed treatment. These findings help establish a rich pool of candidate fungi for further evaluation as novel biological control tools against root-knot nematodes in cotton and other plants.