Cassava root rot disease caused by the fungal pathogens Fusarium solani and Lasiodiplodia theobromae produces severe damages on cassava production. This research was conducted to produce and assess silver nanoparticles (AgNPs) synthesized by Trichoderma harzianum for reducing root rot disease. The results revealed that using the supernatants of T. harzianum on a silver nitrate solution changed it to reddish color at 48 h, indicating the formation of AgNPs. Further characterization was identified using dynamic light scattering (DLS) and scanning electron microscope (SEM). DLS supported that the Z-average size is at 39.79 nm and the mean zeta potential is at − 36.5 mV. SEM revealed the formation of monodispersed spherical shape with a diameter between 60–75 nm. The antibacterial action of AgNPs as an antifungal agent was demonstrated by an observed decrease in the size of the fungal colonies using an increasing concentration of AgNPs until the complete inhibition growth of L. theobromae and F. solani at > 58 µg mL−1 and at ≥ 50 µg mL−1, respectively. At in vitro conditions, the applied AgNPs caused a decrease in the percentage of healthy aerial hyphae of L. theobromae (32.5%) and of F. solani (70.0%) compared to control (100%). The SR-FTIR spectra showed the highest peaks in the first region (3000–2800 cm−1) associated with lipids and fatty acids located at 2962, 2927, and 2854 cm−1 in the AgNPs treated samples. The second region (1700–1450 cm−1) consisting of proteins and peptides revealed the highest peaks at 1658, 1641, and 1548 cm−1 in the AgNPs treated samples. The third region (1300–900 cm−1), which involves nucleic acid, phospholipids, polysaccharides, and carbohydrates, revealed the highest peaks at 1155, 1079, and 1027 cm−1 in the readings from the untreated samples. Finally, the observed root rot severity on cassava roots treated with AgNPs (1.75 ± 0.50) was significantly lower than the control samples (5.00 ± 0.00).