Abstract The root‐knot nematode Meloidogyne incognita is one of the most damaging plant parasitic nematodes in the world. In this study, the effect of cystatin from Amaranthus hypochondriacus (AhCPI) as a potential control agent for M. incognita was explored. In vitro bioassays demonstrated that AhCPI affects the growth and development of eggs and the infectivity of juveniles (J2) of M. incognita , such as mortality and slower development, showing characteristic tissue damage. Mortality levels were quantified by Probit analysis, estimating LC 50 s of 1.4 mg/mL for eggs and 0.028 mg/mL for J2. In planta bioassays showed that infected tomato seedlings treated with 0.056 mg/mL of AhCPI showed a 60% reduction in the number of galls, as compared with untreated J2‐inoculated seedlings. Under greenhouse conditions, three applications of 10 mL of AhCPI (1.4 mg/mL) in the soil around the stem of M. incognita ‐infected tomato plants, reduced the number of galls by 93 ± 8%, as compared to the control M. incognita ‐infected plants. The application of AhCPI to the infected plants increased the yield (10.7%) of harvested tomato fruits, as compared to infected plants. These results show the potential of AhCPI for the control of M. incognita in tomato plants.