Studies have shown that the application of arbuscular mycorrhizal (AM) fungi or exogenous melatonin (MT) can alleviate drought stress and improve plant growth, but the additive effects of both treatments on plants grown under drought stress are largely unknown. In this study, we conducted a pot experiment to investigate the effects of AM inoculation ( Funneliformis mosseae BGC XJ01) and/or MT application on tobacco ( Nicotiana tabacum L. cv. Yuyan No. 6) seedling growth, photosynthetic and chlorophyll fluorescence parameters, antioxidant enzymatic activity, osmotic adjustment substance accumulation, and nutrient uptake under three water conditions (75–80%, 50–55%, and 30–35% of the maximum moisture retention capacity). The results show that applying either the AM inoculant or MT alone significantly increased tobacco seedling growth and decreased the negative effects of drought stress. Furthermore, AM inoculation alone promoted root function (root biomass, root/shoot ratio, root system architecture), facilitated the capture and conversion of solar energy (photosynthetic rate, Φ PSII ), and increased nutrient uptake more effectively than MT. In contrast, exogenous MT application alone was more effective at increasing peroxidase and catalase activity and decreasing H 2 O 2 and MDA accumulation, which in turn enhanced the adaptation of seedlings to drought stress by improving their antioxidant capacity and reducing oxidative damage. Nevertheless, applying exogenous MT significantly enhanced the AM colonization rate under AM inoculation conditions but had no obvious effect on AM colonization under noninoculated conditions. The combined application of AM and MT had an additive effect and produced the largest increases in tobacco seedling growth, photosynthetic ability, antioxidant enzymatic activity, and N, P, and K uptake and the largest decreases in H 2 O 2 and MDA contents of all the treatments. The results suggest that AM inoculation in combination with exogenous MT application may render plants more productive and more tolerant of drought stress.