Extracellular vesicles (EVs) secreted by Lactobacillus species are key mediators of microbial communication, host–microbe interactions, and promising therapeutic agents with potent immunomodulatory and gut-protective properties. These nanoscale vesicles can transport diverse molecular cargo including proteins, lipids, metabolites, and nucleic acids across intestinal barriers. Recent studies have shown that Lactobacillus EVs can replicate many of the probiotic effects of their parental strains, particularly by attenuating host inflammation. Proteomic analyses have revealed a broad spectrum of EV-associated proteins including metabolic enzymes, biogenesis-related factors, and cell wall hydrolases that contribute to their functional activity. In parallel, recent advances in EV engineering such as drug encapsulation, surface modification, and genetic manipulation have expanded their potential as targeted delivery platforms for inflammatory disease treatment. Despite these promising developments, key challenges remain, including the establishment of standardized isolation protocols, consistent and transparent EV characterization, and in-depth mechanistic understanding of EV-host interactions. By addressing current advances in proteomics of Lactobacillus EVs, this review offers a focused evaluation of their composition and biological relevance. It further details methodologies for their isolation and characterization. Their biogenesis, immunological functions, and therapeutic potential are also discussed.