The cuticle is composed of cutin and cuticular waxes, and it is the first protective barrier to abiotic and biotic stresses in fruit. In this study, we analysed the composition of and changes in cuticular waxes during fruit development in nectarine (Prunus persica L. Batsch) cultivars, in parallel with their conductance and their susceptibility to Monilinia laxa. The nectarine waxes were composed of triterpenoids, mostly ursolic and oleanolic acids, phytosterols, and very-long-chain aliphatics. In addition, we detected phenolic compounds that were esterified with sugars or with triterpenoids, which are newly described in cuticular waxes. We quantified 42 compounds and found that they changed markedly during fruit development, with an intense accumulation of triterpenoids during initial fruit growth followed by their decrease at the end of endocarp lignification and a final increase in very-long-chain alkanes and hydroxylated triterpenoids until maturity. The surface conductance and susceptibility to Monilinia decreased sharply at the beginning of endocarp lignification, suggesting that triterpenoid deposition could play a major role in regulating fruit permeability and susceptibility to brown rot. Our results provide new insights into the composition of cuticular waxes of nectarines and their changes during fruit development, opening new avenues of research to explore brown rot resistance factors in stone fruit.