The aim of this study was to determine the influence of mycorrhizal inoculation and sand-peat composition as growth substrate on the biomass, and individual polyphenol concentration of Eclipta prostrata. Mycorrhizal inoculation alters some secondary metabolites of E. prostrata, showing significant differences in polyphenol contents between the treatments. Moreover, varying peat and sand rates, representing different nutrient supplies, had significant impacts on both mycorrhizal colonization and growth responses. Our results highlight that 60/40% (v/v) sand and peat ration is the best for a large-scale cultivation of E. prostrata, moreover supporting the highest total phenolic content. Through high-performance liquid chromatography (HPLC) analysis, nine individual phenolic components were analyzed, including wedelolactone and demethyl-wedelolactone at the highest concentration. Some of the identified compounds, such as 5-o-caffeoylquinic acid, quercetin-3-arabinoside, 4-o-caffeoylquinic acid, and protocatechuic acid have not been reported previously in E. prostrata cultivars. Using hierarchical cluster analysis, multiple groups are represented, suggesting the role of mycorrhizal inoculation, growth substrate, and their interaction on secondary metabolites of E. prostrata. Better understanding of the phenolic composition of E. prostrata and factors influencing it help to identify new industrial applications of this medicinal plant (together with arbuscular mycorrhizal fungi), and moreover, help to develop new strategies for the prevention and treatment of different diseases.