Tomato (Solanum Lycopersicum L.) is an important vegetable crop that belongs to the family Solanaceae. Root-knot nematodes reflect the highly critical economically damaging genera of phytoparasitic nematodes on tomato plants. In this study, the eco-nematicide activity of freshwater green macroalga Cladophora glomerata aqueous extract and their synthesized silver nanoparticles (Ag-NPs) against root-knot nematodes Meloidogyne javanica was investigated on tomato plants. The formation and chemical structure of Ag-NPs was examined. The aqueous extract from C. glomerata was applied against the root-knot nematodes besides the biosynthesized green silver nanoparticles with 100, 75, 50, and 25% (S, S/2, S/3, S/4) concentrations. To investigate the plant response toward the Green Synthesized Silver Nanoparticles (GSNPs) treatment, expression profiling of Phenylalanine Ammonia-Lyase (PAL), Poly Phenol Oxidase (PPO), and Peroxidase (POX) in tomato were examined using Quantitative Real-Time PCR (Q-PCR). The results indicated that GSNPs from C. glomerata exhibited the highest eco-nematicide activity in the laboratory bioassay on egg hatchability and juveniles (J2S) mortality of M. javanica compared with the chemical commercial nematicide Rugby 60%. Also, results showed a significant reduction in galls number, egg masses, females per root system/plant, and mortality of juveniles. The results of PAL and PPO enzyme expression for the control plants remained relatively stable, while the plant inoculated with nematode M. javanica as well as the activity of genes in scope was increased from 14 to 28 Days after Nematode Inoculation (DANI). These activities were improved in inoculated plants and treated with C. glomerata extract and their green syntheses of Ag-NPs and the other plants treated with Rugby 60% (4 mL/L). The greatest activities of the three enzymes were evident after 14 days after the nematode inoculation. It can be concluded that the green synthesized nanoparticles using C. glomerata could be used as potent nematicides against M. javanica which induces the immune system to defend against nematode infection.