Purpose <p>Dissolved organic matter (DOM) was a crucial bioactive component in soil nutrient cycling in new cultivating farmland (NCF). However, the effects of soil conditioners on soil DOM and its microbial community were not completely understood in NCF.</p> Methods <p>The effects of co-application soil conditioners on soil DOM and microbial communities under rice cultivation conditions were investigated through field experiments and three-dimensional fluorescence spectroscopy EEM.</p> Results <p>Results showed that Ca-Mg-Si soil conditioner (SC2) + Earthworm manure + Shrimp shell meal treatments significantly increased SOM (<i>p</i> &lt; 0.05) to 5.52&#xa0;g/kg. And there were significant differences (<i>p</i> &lt; 0.05) in soil DOM components among different soil conditioners. Additionally, the co-application of soil conditioners affect the abundance of <i>SBR1031</i>,<i> Anaeromyxobacter</i> and <i>Haliangium</i>. Fulvic acid-like (FA-like), tyrosine, and other aromatic proteins maintained their dominance of soil DOM in NCF, and this components had a rapid metabolism in the soil and maintained at high concentration in the rice growth stage, which may be regulated by <i>SBR1031</i> and <i>Anaeromyxobacter</i> genus. Moreover, <i>Haliangium</i> may regulate soil DOM transformation to FA-like components. Conclusion: For NCF, the co-application of soil conditioners increased the activity of <i>Haliangium</i> in the short term, and enhanced turnover of FA-like components in soil DOM.</p> Graphical abstract <p></p>