The composition and yield of secondary metabolites of Eucommia ulmoides are important indices of the quality of medicinal materials, and their synthesis and accumulation are affected by both internal and external factors. However, the influential extent and mechanism of these factors on the pharmacologically active ingredients of E. ulmoides are still being elucidated. In this study, we selected 72 of E. ulmoides from 24 regions in China, and investigated the genetic diversity, pharmacologically active ingredients, soil physicochemical properties, and bark bacterial communities, along with the effects of genotypes and environmental factors on pharmacologically active ingredients. Results showed that genetic characteristics among the 24 regions there were no significant different, however, 12 different haplotypes were clearly revealed based on haplotype network analysis. The contents of pinoresinol diglucoside and geniposide acid were mainly affected by different climate and soil physiochemical factors, while aucubin was only sinificantly affected by soil organic matter. All of, the three pharmacologically active ingredients mentioned above were significantly correlated with the dominant genus Lactobacillus, Escherichia, Bacteroides and Faecalibacterium, suggesting that the active ingredients were affected by bark microorganisms. In conclusion, our analysis indicated that the accumulation of pharmacologically active ingredients of E. ulmoides were comprehensively affected genotype, environmental factors (biological and abiotic factors), and their interaction, with genotype had the greatest impact. These results provide important basic information for the future breeding, selection of cultivation conditions and assistance of microbial technology of E. ulmoides.