BACKGROUND: Drought is an abiotic stress that significantly reduces the yield of thyme (Thymus vulgaris). This study investigated how iron oxide nanoparticles (FeNPs), together with symbiotic bacterial (Azospirillum lipoferum) and fungal (Aspergillus oryzae) endophytes, modulate osmotic adjustment, molecular and biochemical mechanisms related to photosynthesis, and drought tolerance mechanisms in thyme. RESULTS: The experiment was evaluated as a factorial experiment in a completely randomized design with three replications. evaluating three treatment factors: four irrigation levels (100%, 75%, 50%, and 25% of field capacity), four FeNPs concentrations (0, 0.5, 1, and 1.5 mg L⁻¹), and three endophyte treatments (control, bacterial (EB), and fungal (EF) inoculation). At 25% FC, EB and spraying with 1 mg L(- 1) FeNPs increased Fv/Fm (maximum quantum efficiency of photosystem II), chlorophyll a, chlorophyll b, and total chlorophyll, carotenoids, relative water content (RWC), and protein levels level protein levels by 18.75%, 10.41%, 31.54%, 18.20%, 14.26%, 35.53%, and 125.22% respectively, compared to the control. At 25% FC, electrolyte leakage (EL) was increased by 47.44% with the combination of EF and 1.5 mg L(- 1) FeNPs. The highest proline accumulation at 25% FC was observed after inoculation with EF and 1 mg L(- 1) FeNPs, resulting in significant increases of 36.36% and 13.04%, respectively, compared to the control. Soluble sugar was remarkably increased by 28.57% under upon treatment with FeNPs (1.5 mg L(- 1) FeNPs). At 25% FC, EB and 1.5 mg L(- 1) FeNPs showed significant reductions of 17.33% and 37.10%, respectively, in malondialdehyde levels compared to control plants. At 50% FC, 1 mg L⁻¹ FeNPs increased Catalase by 15%, peroxidase by 31.25%, and superoxide dismutase by 43.42%, while higher concentrations reduced enzyme activities. Similarly, 1.5 mg L⁻¹ FeNPs and EB inoculation enhanced ascorbate peroxidase by 37.44% and 17.37%, respectively. FeNPs acted as abiotic stressors at low levels but became toxic at higher concentrations. CONCLUSION: Our findings demonstrate that the synergistic application of FeNPs and endophytes significantly enhances drought tolerance in T. vulgaris by optimizing photosynthetic efficiency (Fv/Fm, chlorophyll content) and preserving membrane integrity (RWC, MDA reduction). These results provide a framework for leveraging nano-bio partnerships to improve crop resilience under water scarcity.