In this study, we aimed to evaluate the chemical composition of the essential oil of Chenopodium botrys L. collected from three different parts of Turkey. Additionally, we analyzed the antioxidant activities of the oil samples by using free radical scavenging, phosphomolybdenum, ferrous ion chelating, and reducing power assays as well as their inhibitory activities on acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and tyrosinase. According to the results of GC-FID and GC MS analyses, twenty-seven, twenty-four, and sixteen compounds were identified representing 94.45%, 96.96%, and 94.41% of the oils, respectively. In Afyonkarahisar and Isparta samples, ledol (25.11% and 22.73%), elemol (15.25% and 24.86%), and germacrene D-4-ol (14.83% and 11.27%) were determined as the major compounds, whereas ledol (31.83%), elemol (23.70%), and eudesm-7(11)-en-4-ol (8.94%) were found as the main compounds of Konya sample. The oil of Isparta sample showed the maximum radical scavenging activity in all test systems, except superoxide anion radical scavenging assay. Phosphomolybdenum, ferrous ion chelating, and reducing power assays were resulted in the superiority of Afyonkarahisar sample. In AChE and tyrosinase inhibition assays, the essential oil of C. botrys collected from Konya showed the maximum activity (0.87 mg GALAEs/g oil and 0.82 mg KAEs/g oil, respectively). On the other hand, BChE inhibition assay was resulted in the superiority of Afyonkarahisar sample (1.02 mg GALAEs/g oil). In order to determine the contribution of oil components to the biological activity, correlation coefficients between the compounds and assays were also presented.