<p>Toxin-antitoxin systems (TAs) are genetic modules frequently associated with bacterial stress tolerance, persistence, and pathogenicity. Initially identified on bacterial plasmids and later recognized on chromosomes, the functional roles of chromosomal TAs remain less well understood. This study provides comprehensive in-silico characterization of chromosomally-encoded <i>yefM-yoeB</i>^<i>Xn</i> type II TA module from <i>Xenorhabdus nematophila</i>, including comparative sequence analysis, structural modeling, molecular docking and dynamic stability assessment. Our findings revealed that the YoeB^Xn toxin is well-conserved among gram-negative pathogens, but the YefM^Xn antitoxin is quite variable across species, suggesting significant species-specific regulatory mechanisms. The promoter prediction with putative σ⁷⁰ dependent regulatory region upstream to <i>yefM-yoeB</i>^<i>Xn</i> genes, supported its transcriptional activity. Furthermore, the secondary as well as tertiary models were constructed and validated for their structural integrity. The endoribonuclease activity of YoeB^Xn toxin was predicted by gene ontology analysis, and supported through key interactions in molecular docking and simulation, therefore possibly inhibiting translation. The interaction analysis represented favorable binding of YoeB^Xn toxin to RNA substrate and stable interaction with its cognate YefM^Xn antitoxin. Notably, RNA interacting residues predicted in docking were conserved in the sequence fingerprint and align with experimentally validated catalytic residues reported for <i>E. coli</i>. The MD simulations supported structural stability of YoeB^Xn toxin in both free state and bound to RNA, and indicated reduced flexibility of YefM^Xn upon complex formation, forming stable YefM-YoeB^Xn TA complex. The protein-protein interaction network uncovered potential functional relationship of YefM-YoeB^Xn with various ferric citrate transport proteins, suggesting possible link to iron homeostasis and oxidative stress protection. The YefM-YoeB^Xn TA also suggested potential crosstalk with other toxin-antitoxin partners, based on protein-protein interaction (PPI) network analysis through STRING. Our findings, significantly advances the knowledge of structural as well as functional roles of the chromosomally encoded type-II <i>yefM-yoeB</i>^<i>Xn</i> TA system. Since, our findings are based on computational predictions, experimental validation is required for YoeB^Xn toxicity, its neutralization, RNA specificity, and physiological roles during stress adaptation.</p>