The effects of Bacillus velezensis (Bv) PGPR strains amended with orange peel powder (OPP) on the mortality of second-stage juveniles (J2) of root-knot nematode (RKN) (Meloidogyne incognita) were investigated in vitro. We observed that Bv strains grown on OPP strongly expressed many secondary metabolites, and their metabolome was distinct from that of Bv strains grown on glucose; therefore, we hypothesized that Bv strains grown on OPP would have enhanced expression of metabolites that increase the mortality rate of J2 compared to Bv strains grown on glucose. The treatments included inoculation of J2 with cell-free supernatants (CFS) from Bv strains grown on OPP or glucose, J2 with OPP and non-inoculated controls. The results showed that CFS from Bv strains AP136 and AP203 grown on OPP significantly increased J2 mortality compared to other treatments (P < 0.05). The J2 mortality rate increased with OPP-grown strain AP203 (85.2%) or AP136 (75.2%) compared to these strains grown on glucose (52.3% or 49.1%, respectively) or with the presence of OPP (44.2%) or the non-inoculated control (4.5%). The unexpected increase in J2 mortality in the presence of OPP alone may be due to effects of OPP on nematode-associated microbiota. A draft genome of Bv AP136 was used to predict the presence of eight different biosynthetic gene clusters, some of which do not correspond to known metabolites. Ongoing LC-MS analyses are being conducted to determine the effects of growth substrate (glucose or OPP) on the expression of Bv secondary metabolite(s) responsible for death of the J2 RKN.