【Objective】 The objective of this study was to investigate the impact of Streptomyces pactum Act12 broth on growth and physiological characteristics of Medicago sativa seedlings under petroleum stress.【Methods】 The effects of different concentrations (0, 1%, 10%, and 100%) of Act12 broth under a petroleum stress level of 3% (W/W) were examined on various parameters including seedling growth, root morphology, photosynthetic characteristics, osmoregulatory substance content, and antioxidant capacity in Medicago sativa. 【Results】 The results demonstrated that exposure to 3% petroleum stress significantly hindered photosynthesis and biomass accumulation of Medicago sativa seedlings, when compared to the control group (CK). Moreover, it resulted in an elevation in malondialdehyde levels, a decrease in soluble sugar and soluble protein content, as well as alterations in the activities of superoxide dismutase (SOD) and peroxidase (POD). Compared to the absence of Act12 broth, solutions with low and medium concentrations (1% and 10%) effectively mitigated the toxic effects of petroleum stress, with 10% Act12 broth exhibiting the most significant effect. The seedlings treated with Act12 broth exhibited significant enhancements in plant height, above-ground dry weight, underground dry weight, stem tolerance index, root tolerance index, total root length, root surface area, root volume, and number of root tips. Additionally, the levels of MDA decreased by 42.49% and 56.45%, soluble sugar increased by 79.25% and 89.83%, while soluble protein increased by 167.63% and 256.15% in the above-ground and underground parts respectively. Moreover, SOD activity increased by 35.81% while POD activity of roots increased by 57.33%. 【Conclusion】 The fermented liquid with low/medium concentration effectively modulated the activity of antioxidant enzymes and osmotic regulation ability of seedlings, resulting in reduced MDA content, enhanced photosynthesis ability, increased biomass, improved resistance, and alleviated the inhibition of oil stress on seedling growth.