In this current study, Vitex agnus-castus seed ethanol extracts were analyzed for their phytochemical component content, anticholinergic and antioxidant activities, and antibacterial properties. The phenolic compound composition of these seeds was determined by using LC/MS/MS. Antioxidant activity of the seeds was examined by the DPPH, ABTS, Fe 3 + -Fe 2 + reducing, and CUPRAC. Also, the anticholinergic activity was measured by the inhibition of acetylcholinesterase (AChE). The antibacterial activity was performed by disc diffusion and minimum inhibitory concentration methods. The main phenolic compound was vanillic acid (22812.05 μg/L) and followed by luteolin, fumaric acid, quercetin, caffeic acid, 4-hydroxybenzoic acid, salicylic acid, kaempferol, butein, ellagic acid, resveratrol, catechin hydrate, phloridzin dehydrate, naringenin, respectively. The DPPH free radical scavenging value of ethanol extract of plant seeds was 9.41 %, while the ABTS radical scavenging activity was determined as 12.66 %. The ethanol extract of the seeds exhibited antibacterial activity on Escherichia coli, Staphylococcus aureus, and Salmonella Typhimurium, differently. S. aureus was found to be more susceptible to the extract than other bacteria. Also, the inhibition effect of seed ethanolic extract on the AChE with IC 50 values were 36.34 � 5.6 μg/mL. From the results, V. agnus-castus seed can be suggested as a promising natural antioxidant and antibacterial candidate for the preservation of foods.