The use of biofertilizers as an alternative to mineral materials has been widely developed to reach optimum productivity in medicinal plants. The present study aimed to evaluate the effects Arbuscular mycorrhizal fungi (AMF) inoculation (Rhizophagus intraradices), plant growth-promoting rhizobacteria (PGPR: Pseudomonas putida and Azospirillum lipoferum), and mineral fertilizer (NPK) on the plant growth, essential oil (EO) content, EO composition, and antioxidant activity of Satureja macrantha L on a loamy soil in semi-arid area of Iran. The results revealed that AMF, NPK, A. lipoferum, and P. putida increased dry weight yield (DWY) by 26%, 24%, 17%, and 22%, respectively, compared with control in August 2018. Mycorrhizal plants had higher chlorophyll (Chl) a + b and total phenolic content (TPC) in comparison with no amendment application. Total flavonoid content (TFC) in plants supplied with AMF and NPK was higher than in other treated and untreated plants. In the second-year plants, the highest EO content and yield were observed in plants supplied with AMF followed by P. putida, A. lipoferum, and NPK, respectively. The GC/MS analysis showed that monoterpenes characterized the main EO profile of S. macrantha L., including p-cymene (22.14%–33.57%), γ‑terpinene (22.26%–36.28%), thymol (0.11%–31.23%), and carvacrol (0.36%–36.56%). According to agglomerative hierarchical clustering (AHC), AMF and NPK were placed in the same cluster; therefore, AMF can be selected as a proper alternative to chemical fertilizer for improving plant yield and EO yield. By assisting biofertilizers in reaching their maximum performance of S. macrantha L. with significant pharmacological potential, the results may be useful for its development in semiarid regions.