With the aim to establish a novel nano-formulation with a high nematocidal activity and safety for human as well as the ecosystem, in this work, lipid based nanoemulsions (LNs) co-encapsulating chili oil, cinnamon oil and neem oil (NaTri) was prepared by a combined method of homogenization and sonication. The mean particle size, polydispersity index (PDI), zeta potential of the prepared NaTri was 156.7 nm, 0.193 and −36.8 mV, respectively. NaTri showed a fast penetration into nematodes as well as plant roots, and it did not show any phytotoxicity. The presence of chili oil has showed a synergistic effect with cinnamon oil and neem oil resulting in a high nematocidal activity of NaTri. Particularly, its in vitro nematocidal activity at a 300 times dilution was 82.3%, while its ex vitro nematocidal activity at a 300 times dilution was more than 75% for the sprinkler irrigation and 91.3% for the drip irrigation. This novel nano-formulation shows great potential of wide application in sustainable horticulture to protect plants from the adverse effects of nematodes.