Peroxisomal beta-oxidation (p beta o) is a highly conserved fat metabolism pathway involved in the biosynthesis of diverse signaling molecules in animals and plants. In Caenorhabditis elegans, p beta o is required for the biosynthesis of the ascarosides, signaling molecules that control development, lifespan, and behavior in this model organism. Via comparative mass spectrometric analysis of p beta o mutants and wildtype, we show that p beta o in C. elegans and the satellite model P. pacificus contributes to life stage-specific biosynthesis of several hundred previously unknown metabolites. The p beta o-dependent portion of the metabolome is unexpectedly diverse, e.g., intersecting with nucleoside and neurotransmitter metabolism. Cell type-specific restoration of p beta o in p beta o-defective mutants further revealed that p beta o-dependent submetabolomes differ between tissues. These results suggest that interactions of fat, nucleoside, and other primary metabolism pathways can generate structural diversity reminiscent of that arising from combinatorial strategies in microbial natural product biosynthesis.