Plant-parasitic nematodes (PPN) are the most damaging plant pathogens all over the world. Root-knot nematodes (RKNs), considered the most important phytonematodes globally, cause significant agricultural losses. Despite the availability of various strategies to manage these pathogenic agents, excessive use of nematicides poses a threat to human health and the environment. Compounds derived from plant sources are proposed as an alternative to new biocides, potentially offering advantages over synthetic components. Several species within the Fabaceae family, including those within the Arachis genus, have demonstrated potential as sources of nematotoxic compounds. As part of a research program aimed at exploring bioactive compounds and valorizing germplasm banks, this study evaluated the nematicidal and nematostatic effects of aqueous crude extracts (ACEs) obtained from nine Arachis species sourced from the Embrapa Active Germplasm Bank against M. incognita’s second-stage juveniles (J2). The results indicate that Arachis stenosperma (ACE1) has promising nematocidal potential, with effectiveness exceeding 95% on dead nematodes for doses above 0.5 mg/mL. ACE1 has also demonstrated thermostability and lower harmful effects on bovine cells. This research provides a fresh outlook on the promising use of preserved germplasms to enhance Germplasm Storage Bank’s value, given the underexplored potential of these biological assets.