Xenorhabdus is an entomopathogenic bacterium involved in a mutualistic relationship with Steinernema nematodes. Xenorhabdus produces a multitude of specialized metabolites by Non-Ribosomal Peptide Synthetases (NRPSs) pathways to mediate bacterial-nematode-insect interactions. PAX cyclolipopeptides are a family of NRP-type molecules whose ecological role remains poorly understood. In this study, the pleiotropic role of PAX peptides in the life cycle of Xenorhabdus nematophila has been investigated. By mass spectrometry analysis, we first demonstrated that PAX peptides were detected from the pathogenic stage up to the necrotrophic stage. We discovered that the bromothymol blue adsorption phenotype historically used to discriminate Xenorhabdus variants was associated with the presence of PAX peptides. We found that PAX peptides were positively involved in biofilm formation and negatively involved in swimming motility. PAX peptides were also shown to promote in vivo the production of infective Steinernema juveniles, suggesting their involvement in the mutualistic relationship between Xenorhabdus and its nematode partner. Finally, we showed that the paxTABC cluster as well as PAX peptide production was conserved across the whole Xenorhabdus genus except in Xenorhabdus poinarii and Xenorhabdus ishibashii. This work has revealed multiple new ecological roles for NRP-type peptides.