Extracellular vesicles (EVs) and HIV-1 Gag virus-like particles (VLPs) represent promising platforms for therapeutic delivery and vaccine development. However, their molecular cargo may critically influence their biological properties and clinical applicability. In this study, a multi-omic approach combining transcriptomic and lipidomic analyses was applied to characterize and compare the RNA and lipid composition of EVs and VLPs produced in human embryonic kidney (HEK293) cells. Transcriptomic analysis revealed broader RNA cargo shifts in VLPs, including enrichment of mRNAs related to protein degradation, RNA regulation, and translation, while EVs produced under cell growth exhibited upregulation of histone-encoding mRNAs. Tumor suppressor, oncogenic microRNAs (miRNAs) and adenoviral mRNAs were identified in both EVs and VLPs, with the latter enriched in VLPs. This emphasizes the need for careful evaluation in therapeutic applications. VLPs showed a lower proportion of mature miRNAs compared to EVs, suggesting differences in miRNA processing or loading. Lipidomic profiling showed reduced total lipid content in VLPs, but enrichment of lipid species associated with enhanced membrane stability. Overall, these findings highlight key molecular differences between EVs and VLPs and provide valuable insights for optimizing vesicle-based strategies for RNA delivery and vaccine development.