Rhabdomyolysis-associated acute kidney injury is driven by myoglobin-mediated oxidative stress, inflammation, mitochondrial impairment, and tubular cell death. This study evaluated the nephroprotective activity of green-synthesized Withania somnifera-functionalized selenium nanoparticles (Ws-SeNPs) in glycerol-induced renal injury and compared their efficacy with native W. somnifera extract and sodium selenite. The chemical profile of the plant extract was characterized by LC–MS/MS, and Ws-SeNPs were evaluated using dynamic light scattering, zeta potential analysis, transmission electron microscopy, and FTIR spectroscopy. Thirty-five male rats were assigned to Control, ARF, ARF & Ws, ARF & selenium, and ARF & Ws-SeNPs. ARF was induced by intramuscular injection of 50% glycerol. Glycerol administration induced marked skeletal muscle injury, renal dysfunction, tubular damage, oxidative stress, inflammation, mitochondrial dysregulation, pyroptosis, apoptosis, and histopathological alterations. Both Ws and sodium selenite provided partial protection, whereas Ws-SeNPs produced the greatest improvement in renal function and tissue architecture. Their protective effect was associated with restoration of Nrf2-dependent antioxidant defenses, suppression of NF-κB/NLRP3/GSDMD-associated inflammatory and pyroptotic signaling, preservation of mitochondrial regulatory pathways, and attenuation of apoptosis. These findings indicate that Ws-SeNPs provide multi-target protection against glycerol-induced rhabdomyolysis-associated renal injury and may represent a promising phytochemical-based selenium nanoformulation for further preclinical investigation.