The nematicidal efficacy of milbemectin and its commercial formulate Milbeknock® on (i) egg hatching, (ii) juvenile motility and (iii) infective capacity of root-knot nematodes was evaluated in vitro and in planta assays. Serial dilutions of pure milbemectin were tested against nematode eggs and juveniles and lethal concentrations LC50 and LC90 calculated. Exposure of egg masses to milbemectin at a concentration of 30 μg/mL for 72 h reduced egg hatching by 52%. The increase in exposure time to 240 h did not increase the egg hatching inhibition at the highest concentration 30 μg/mL (53%) but reduced egg hatching at 15 and 7 μg/mL by 35 and 24%, respectively, when compared to untreated controls. The inhibitory effect of milbemectin on juvenile motility ranged from 41 to 87% depending on its concentration, and this effect was persistent after rinsing the juveniles in water. The probabilistic dose-response model indicated that lethal concentrations of milbemectin for juvenile motility were LC50: 7.4 μg/mL and LC90: 29.9 μg/mL. The pre-plant application of Milbeknock® to soils infested with the nematode reduced its infective capacity by 98-99% compared to untreated soils in pot experiments. Milbeknock® reduced nematode soil population densities by 50-60% in natural infestations under field conditions. Milbemectin shows a high level of efficacy against root-knot nematodes as it reduces egg hatching, persistently immobilizes nematode juveniles, and reduces tomato root infection.