Cyclocarya paliurus (Batal.) Iljinsk is an important medicinal plant for treating chronic diseases, but it is difficult to obtain high yields when growing on low-fertility soil. Inoculation with soil beneficial microorganism has suggested an effective means of stimulating plant growth and secondary metabolite production, but effect on plant performance when competing degraded field condition remains unclear. We combined controlled laboratory experiments with field trials to investigate the effects of co-inoculation with phyto-stimulatory strains (Azospirillum brasilense and Pseudomonas fluorescens) and nutrient-enhancing strains (Bacillus megaterium and Azotobacter chroococcum). Bacteria were applied with organic fertilizer at different fertilizer levels, and we tracked effects on soil nutrient availability as well as C. paliurus morphological traits, photosynthesis, growth and bioactive compounds during cultivation on barren land. Amendment of beneficial microbes with organic fertilizer enhanced the soil nutrient availability with high fertilizer showing greatest stimulation under controlled conditions, with the medium fertilizer giving best results in improving plant performance in the field. All fertilization regimes expanded the 3D root architecture, and bacterial additions increased the proportion of lateral roots compared to a single organic fertilizer treatment, which led to higher nutrient uptake. Inoculations at medium fertilizing level modified the root system and increased the photosynthesis rate, nutrient acquisition and plant growth. The co-inoculation with B. megaterium and P. fluorescens at medium fertilizer level stimulated the accumulation of flavonoids and polysaccharides, while co-inoculation with A. chroococcum and A. brasilense at low fertilizing level facilitated the production of flavonoids and triterpenoids. The biosynthesis of secondary metabolites exhibited strong correlations with leaf C/N and C/P ratios. Thus, manipulation of bioactive compounds in C. paliurus leaves can be affected by internal nutrient balance, which is associated with reformed root system morphology that modulated by bacterial inoculation.