Bell and chili peppers are important vegetable and spice commodities worldwide. Significant yield reductions have been attributed to damage caused by root-knot nematodes (RKNs; Meloidogyne spp.). This study addresses the need for developing pepper varieties that have high resistance to RKN, which is now of increasing importance due to restrictions on the use of fumigant nematicides. Our goal is to provide a nonchemical alternative to sustain commercial pepper production in Florida, which is a major producer of peppers in the United States. We evaluated ‘UFRJ107(6)A3’, an advanced inbred line developed from a cross between Capsicum annuum L. ‘Jalapeno’ and ‘Round of Hungary’, for resistance against the nematode in comparison with the parental and three other Capsicum cultivars, namely, C. annuum ‘Charleston Belle’, ‘California Wonder’, and C. chinense Jacq. ‘Datil’ in two separate growth chamber experiments. Based on egg mass indices and reproduction factors, ‘UFRJ107(6)A3’ was significantly more resistant to M. incognita compared with the other five cultivars. When tested with five RKN species, ‘UFRJ107(6)A3’ showed comparably high levels of resistance to M. arenaria and M. floridensis as ‘California Wonder’ based on the nematode reproduction factor. In ‘UFRJ107(6)A3’, however, there were no detectable M. arenaria egg masses, and M. incognita reproduction was minimal compared with that of ‘California Wonder’; both cultivars supported the reproduction of M. enterolobii and M. javanica, although the reproduction factors of M. enterolobii were ≈10-fold higher than M. javanica. To characterize the mechanism of high resistance to M. incognita in ‘UFRJ107(6)A3’, we examined the extent to which infective second-stage juveniles (J2s) were able to penetrate its roots in comparison with the susceptible ‘California Wonder’ and ‘Datil’ in two independent experiments; we conducted RKN root penetration assays with a single plant in a pot and two plants in a single-pot choice test using ‘Datil’ and ‘California Wonder’, respectively, as susceptible standards. In both assays, M. incognita J2s were absent in the roots of ‘UFRJ107(6)A3’ 7 days after inoculation but were present in the susceptible cultivars, indicating that resistance has an effect at the root invasion stage. In growth chamber experiments, at constant temperatures of 28 and 30 °C, ‘UFRJ107(6)A3’ exhibited M. incognita resistance superior to its parents and to the standard resistant bell pepper ‘Charleston Belle’, thus offering the potential to enhance specialty pepper production and for use as a nematode-resistant rootstock for commercial bell peppers.