The extracts of Glaucium grandiflorum have been used to treat neurodegenerative diseases. Nonetheless, no former study has investigated whether the alkaloid extracts of G. grandiflorum have antioxidative effects against oxidative stress. The aim of the present study was to determine the antioxidative effects of the alkaloid extracts of G. grandiflorum with a variety of targets and probable mechanisms. First, we used spectrophotometry to investigate alkaloid extracts with respect to their alkaloid amounts. Then, we determined the alkaloid extracts’ impact on thiol/disulfide homeostasis, total oxidant status/total antioxidant status/oxidative stress index, and antioxidant enzyme activities. Finally, the effects of alkaloid extracts on the genes in the NRF2-KEAP1 pathway were determined via qRT-PCR. We conducted molecular docking analyses to determine the potential binding of isoquinoline alkaloids found within the alkaloid extracts with target proteins. We observed the best results from chloroform alkaloid extract and methanol alkaloid extract. Chloroform alkaloid extract was prominent in DPPH radical scavenging and metal ions chelating, and methanol alkaloid extract showed significant hydroxyl radical scavenging, lipid peroxidation, and superoxide anion radical scavenging activity. Alkaloid extract groups substantially increased in total thiol activity, native thiol activity, disulfide activity, total antioxidant status level, antioxidant enzyme levels, and gene expression levels (GCLC, HO-1, NRF2, and NQO1) compared to the H2O2 group. Also, alkaloid extract groups led to a significant drop in total oxidant status level, oxidative stress index level, and KEAP1 gene expression level relative to the H2O2 group. According to our study results, oxidative stress brought about by H2O2 was regulated by alkaloid extracts. As a result, a phytochemical-based therapeutic that regulates H2O2-induced oxidative stress was brought to the neurochemical field.