Morus alba root bark (MA) has been long used in traditional Chinese medicine for treating diabetes, inflammation, hypertension, hypoglycemia, and has shown neuroprotective potential, particularly in Alzheimer’s disease. Glaucoma is a progressive neurodegenerative disease leading irreversible blindness by damaging retinal ganglion cells (RGCs), highlighting the need for new treatments. In this study, we evaluated the neuroprotective activity of the ethyl acetate fraction (MAEA) in R28 retinal cells exposed to glutamate-induced toxicity. From MAEA, 23 compounds were isolated, including eight new structures (1–8) identified by NMR and HRESIMS. Screening revealed four compounds (9, 11, 18, and 19) with significant protective effects. Among them, compound 19 (Albasin B, AB) showed the strongest activity, reducing reactive oxygen species levels and cell death at low concentrations. AB was further tested in a mouse model of NMDA-induced retinal degeneration. Optical coherence tomography and hematoxylin and eosin staining showed that AB preserved retinal thickness and reduced RGC loss. Western blot analysis confirmed that AB decreased cleaved caspase-3 and BAX expression in NMDA-treated retinas. These findings indicate that AB, isolated from Morus alba root bark, significantly protects RGCs from excitotoxicity and represents a promising therapeutic candidate for glaucoma treatment.