BACKGROUND: Enhancing the content of essential oils and valuable secondary metabolites is a primary goal for medicinal plant breeders. In this study, the effects of Thymbra spicata extract at concentrations of 0% (C), 10% (TS1), and 20% (TS2), along with mycorrhizal fungus (MF) biofertilizer at a rate of 50 g/2.5 kg of soil, were evaluated on the growth, photosynthetic pigments, relative water content (RWC), proline, protein, malondialdehyde (MDA), catalase (CAT), phenylalanine ammonia-lyase (PAL), and essential oil content and composition of Rosmarinus officinalis L. under varying salinity stress levels of 0 mM (S0), 100 mM (S1), and 200 mM (S2) NaCl. The experiment was conducted as a factorial study within a completely randomized design, with three replications. RESULTS: As salinity stress increased, the yield and growth characteristics of the plants declined. However, the applied treatments effectively mitigated the negative effects of salinity. The highest chlorophyll a, b, and total chlorophyll contents were observed in the TS2 + MF treatment under nonsaline conditions. Under S2 salinity stress, carotenoid and anthocyanin contents increased by 38.29% and 11.11%, respectively, with the use of TS2 + MF. Under S1 stress conditions, the proline and soluble sugar content increased by 268% and 44%, respectively, in the MF treatment. Essential oil content was enhanced by 80.43% with the TS2 + MF treatment under S1 stress. Essential oil analysis showed significant increases in camphene (9.71%), β-pinene (43.75%), α-phellandrene (13.3%), geranyl acetate (156%), cineole (21.39%), and β-linalool (5.12%) in the TS2 + MF treatment compared to the control under S1 stress conditions. CONCLUSIONS: Among all the treatments, the combined application of TS2 and MF proved to be the most effective in enhancing the morphophysiological and biochemical characteristics of rosemary plants. This treatment not only boosted the production of essential oils and secondary metabolites but also mitigated the detrimental effects of salinity stress. Therefore, it is recommended as a beneficial agricultural practice for improving the productivity and quality of rosemary plants under salinity stress.