Background <p>Acetylcholinesterase (AChE) and toll-like receptor 4 (TLR4) are particularly involved in Alzheimer’s disease (AD), a major cause of morbidity and mortality worldwide. AChE and TLR4 inhibitions play crucial roles in treating neurodegenerative disorders. <i>Pulmonaria officinalis</i> (PO; lungwort) has shown in vitro antioxidant, anti-inflammatory, and AChE inhibitory activities. However in vivo biological properties are still insufficiently explored.</p> Methods <p>This study aimed to characterize the metabolomic profile and antioxidant activities of different PO fractions, specifically butanol (BuOH) and ethyl acetate (EtOAc), using colorimetric, spectrophotometric, and chromatographic methods. In addition, studying their neuroprotective effect in lipopolysaccharide (LPS)-triggered Alzheimer’s in mice. The qualitative phytochemical analysis revealed that the fractions of PO contain alkaloids, glycosides, phenols, flavonoids, terpenoids, and tannins.</p> Results <p>The lungwort plant was identified with 36 active compounds in the two fractions. Extractions of EtOAc and BuOH lungworts were rich in polyphenolic compounds identified by HPLC and HPLC-mass spectrometry (HPLC-MS) analysis. Regarding phenolic, tannin, and flavonoid content, PO showed that the EtOAc and BuOH fractions (ABTS•⁺ IC₅₀: 29.64 ± 0.33, 37.46 ± 0.38 and DPPH•⁻ IC₅₀: 43.79 ± 0.66, 38.82 ± 0.35, respectively) were more significant than the other fractions. In the in vivo experiment, mice were randomly divided into four groups, each comprising eight animals. Group I was designated as the normal control, whereas Group II was administered lipopolysaccharide (LPS; 250&#xa0;µg/kg, intraperitoneally). Groups III and IV were administered PO butanol and ethyl acetate fractions (400&#xa0;mg/kg, orally) for seven consecutive days along with LPS treatment. PO fractions ameliorated cognitive dysfunction, inhibited neuroinflammatory cytokines IL-1β and TLR4, alleviated neurodegeneration and elevated the memory via reducing significantly AChE by 73% and 72% and amyloid beta by 37% and 56%, as compared with LPS-treated mice.</p> Conclusions <p><i>P. officinalis</i> was investigated in vivo for the first time and has a neuroprotective effect against AD, which may be attributed to the presence of the identified metabolites in this study. The observed effects support the biological relevance of <i>P. officinalis</i> within the experimental models evaluated.</p> Graphical abstract <p></p>