A decline in mitochondrial functions associated with ageing is the key factor of free radical generation which contributes to age-related pathologies. Protecting healthy functional mitochondrial networks with antioxidants is critical in promoting healthy ageing. This study aimed to investigate the protective effect of ergothioneine (EGT)-rich Lentinula edodes extract (LE-ETH) against tert-butyl hydroperoxide (t-BHP) assaulted senescent HT22 cells. Mitochondrial function was evaluated by measuring mitochondrial membrane potential (MMP), ATP levels and mitochondrial toxicity. The protective mechanisms were elucidated via the exploration of antioxidant and mitochondrial biogenesis signalling pathways. Our results revealed that a low dose of t-BHP increases mitochondrial toxicity. The pretreatment with 100 µg/mL of LE-ETH and the equimolar concentration of EGT for 8 h significantly improve the mitochondrial function and reduced inflammation. Through gene expression studies, we demonstrated that pretreatment of LE-ETH significantly improves the antioxidant and mitochondrial biogenesis pathway via Nrf2 signaling axis. However, the downstream genes of the mitochondrial biogenesis pathway were unaffected by equimolar EGT concentration. Gas chromatography-mass spectrum (GC-MS) analysis was carried out to identify the bioactive compounds that are present in LE-ETH extract which contributed to its efficacy in improving the mitochondrial functions. A total of 23 compounds consisting of phenols, fatty acids, and sterols were identified in the ethanolic extract. Pentanoic acid was the major compound identified in LE-ETH. These findings demonstrated that EGT-rich L.edodes mushroom is a potential neuroprotective agent which could serve as a potential therapeutic strategy for the preservation of mitochondrial functions in healthy ageing explorations.