This study evaluated secondary metabolites, essential oil compositions, and antioxidant activity in four medicinal plants that originated in Isfahan rangelands. The species were Astragalus verus, Astragalus adscendens, Daphne mucronata, and Phlomis olivieri. Thirty‐two genotypes of these species were evaluated for different biochemical traits. Based on the evaluation of EO compounds, GC‐MS analysis revealed the total number of identified compounds. These compounds were 25, 22, 12, and 22 for A. adscendens, A. verus, D. mucronata, and P. olivieri, respectively. The dominant compounds were phthalate (59.88%) in A. adscendens, phytol (38.02%) in A. verus, hexanoic acid (32.05%) in D. mucronata and β‐cubebene (30.94%) in P. olivieri. Phytochemical analysis showed that D. mucronata, A. adscendens, and P. olivieri had the highest total phenolic content (TPC) (18.24 mg gallic acid equivalent/g dry weight), total flavonoids (5.57 mg QE/g DW), and total anthocyanin content (0.23 mg/g DW), respectively. The highest total chlorophyll (0.27 mg/g DW), total carotenoids (0.03 mg/g DW), and antioxidant activity (71.36 %) were observed in A. adscendens, A. adscendens and A. verus, respectively. The data revealed that superior genotypes from these species are rich in natural antioxidants and bioactive compounds. Thus, they can be used in ethno pharmacological fields and industrial applications.