The novelty of this study is to present a green method for the synthesis of Fe3O4 nanoparticles (Fe3O4NPs) using the plant extract of Anacardium occidentale. Fe3O4NPs were synthesized by a simple, cost-effective and environmentally friendly method. Various spectroscopic techniques confirmed the successful synthesis of Fe3O4NPs. UV–Vis spectrum analysis confirmed the synthesis of Fe3O4NPs at absorption of 310 nm. FTIR analysis confirmed the presence of several important functional groups and XRD analysis showed that Fe3O4NPs have a crystalline structure. FESEM and HR-TEM analyses showed that the particles have a spherical shape with average particle size of 15.82 nm. Furthermore, Fe3O4NPs indicate that they have excellent antioxidant activity of DPPH (64.92 ± 1.17 %) and ABTS (73.67 ± 1.13 %). In antibacterial activity, Fe3O4NPs exhibited the highest ZOI against Escherichia coli (24.58 ± 1.29 mm) and Staphylococcus aureus (23.74 ± 1.24 mm). At a concentration of 100 mg/mL, Fe3O4NPs showed the most anti-inflammatory activity against COX-1 (70.35 ± 0.89) and COX-2 (76.48 ± 0.93). In addition, Fe3O4NPs exhibited excellent enzymatic inhibitory activity of α-amylase (63.47 ± 0.97 %) and α-glucosidase (70.58 ± 1.14 %). The anti-cholinesterase activity of Fe3O4NPs was demonstrated by the enzyme inhibition of AChE (76.19 ± 1.10 %) and BuChE (84.91 ± 1.14 %). Cytotoxicity study showed that Fe3O4NPs at 100 µg/mL significantly reduced cell viability (24.78 ± 0.56 %) of HepG2 liver cancer cells. Molecular docking studies confirm that strong binding affinity to AChE and BuChE proteins. Overall, this study conclude that the synthesized Fe3O4NPs have considerable efficacy for various biological applications. © 2025 Elsevier B.V., All rights reserved.