Biological control of gastrointestinal nematodes is an alternative to chemical parasitic treatments. Nematophagous fungi can be developed as biological control products for gastrointestinal nematodes due to their capacity to produce traps that capture nematodes. Nevertheless, the lack of knowledge regarding the mass production of chlamydospores limits the production of biological products as alternatives to traditional antihelminthics. This study evaluated single and bi-phasic fermentation systems with three fermentation subsystems (entire grain, milled grain with low water content and milled grain with high water content) and four substrates (rice, barley, corn, and wheat), on the chlamydospores production of the nematophagous fungus Duddingtonia flagrans. The highest chlamydospores concentration was achieved using a bi-phasic fermentation system with rice as a substrate with a solid fermentation subsystem, which produced 2.01 × 108 chlamydospores g−1 after 14 days of fermentation.