Meloidogyne incognita (root-knot nematode) is a devastating soil-borne pathogen which can infect almost all cultivated plants around the globe, expediting huge pecuniary losses. The purpose of current study was to use the aqueous root extract of Glycyrrhiza glabra for synthesizing silver nanoparticles (GRAgNPs) and assess their nematicidal potential against M. incognita by in vitro methods, including hatching inhibition and mortality assays. The active uptake of FITC labeled GRAgNPs by the nematode and their effect on the expression of selected genes involved in oxidative stress and DNA damage repair were also studied. An HRTEM micrograph confirmed their spherical morphology with sizes ranging from 9.61 nm to 34.735 nm. Complete inhibition of egg-hatching was observed after 48 h of treatment with as low as 10.0 ppm of GRAgNPs. In addition, 100% mortality was recorded at the lowest dose of 6.0 ppm, after 12 h of treatment. The LC-50 for GRAgNPs was found to be 0.805 ± 0.177 ppm at p < 0.0001, R2 = 0.9930, and α = 0.05. The expression of targeted genes (skn-1, mev-1, sod-3, dhs-23, cyp-450, xpa, cpr-1, gst-n, and ugt) was significantly enhanced (1.09−2.79 folds), at 1.0 ppm (α = 0.05, 95% CI) GRAgNPs treatment. In conclusion, GRAgNPs performed efficaciously and considerably in contrast to chemical nematicide and commercial silver nanoparticles (CAgNPs) and might be used as a promising alternative as relatively lower concentration and short exposure time were enough to cause higher mortality and nanotoxicity in nematodes.