The application of the bionematicides derived from microorganisms and their secondary metabolites represents a promising strategy for managing root-knot nematodes. In this study, a nematicidal compound, cis-3-indoleacrylic acid, was isolated from Streptomyces youssoufiensis YMF3.862. This compound caused Meloidogyne incognita juveniles to have swollen bodies with apparent cracks on the cuticle surface. The LC50 value of cis-3-indoleacrylic acid against juveniles was 16.31 μg/mL 24 h of post-treatment. Cis-3-indoleacrylic acid at 20 μg/mL significantly inhibited V-ATPase expression and remarkably decreased enzyme activity by 84.41%. As an inhibitor of V-ATPase, cis-3-indoleacrylic acid caused significant H+ accumulation in nematode bodies, resulting in lower intracellular pH values and higher extracellular pH values of M. incognita. Application of 50 μg/mL cis-3-indoleacrylic acid generated a 71.06% control efficiency against M. incognita on tomatoes. The combination results of this study indicated that cis-3-indoleacrylic acid can be developed as a natural nematicide for controlling M. incognita.