Purpose <p>Application of organic substrates to rehabilitate mine tailings is a cost-effective and commonly employed strategy. Although the remediation efficiency of various amendments has been investigated, the effectiveness of organic substrate in enhancing the microbiome in iron ore mine tailings remains unknown.</p> Methods <p>This study hypothesised that organic substrate could enhance the root growth of perennial ryegrass (<i>Lolium perenne</i>) over 60 days under controlled glasshouse conditions in two distinct iron ore mine tailings (A and B). The impacts of organic substrate on physicochemical properties of tailings, root architecture, bacterial communities, and the abundance of C- and N-cycling genes were investigated.</p> Results <p>In contrast to control, tailings treated with organic substrate at 75% (w/w), the concentrations of diethylenetriamine-pentaacetic acid (DTPA)-extractable Cr and Zn increased (38% and 54%), in tailings A, but remained the same in tailings B, whereas DTPA-extractable Cu and Fe increased (3-fold) and Mn (2-fold) decreased regardless of tailings type. Compared with the control, organic substrate application at 75% increased perennial ryegrass root biomass, length, and average root diameter in both tailings. Further, organic substrate application at 75% showed a substantial increased in both microbial biomass carbon and microbial respiration. The relative abundance of Proteobacteria and Bacteroidetes decreased, while that of Actinobacteria increased, with an increasing rate of organic substrate (75%). Amylose degradation was the least prominent process (<i>SusB</i>) in C cycling, whereas denitrification (<i>narG</i>,<i> nirK</i>,<i> nosZ</i>,<i> nrfA</i>) was the most prominent process in N cycling in both tailings.</p> Conclusion <p>Overall, compared with tailings A, tailings B provided a better environment for plant growth and microbiome abundance and activity, with the addition of organic substrate further improving tailings B. This study highlights the potential of organic substrate use in rehabilitation of iron ore mine tailings via enhancing their biological attributes; however, further research is required to validate these findings under field conditions.</p>