Microcystin (MC) contamination of surface waters threatens ecosystems and public health. Nature-based solutions such as Multi-Soil-Layering (MSL) systems have been used for MC remediation. However, the biological mechanisms controlling MC degradation remain unclear. The present study investigates microbial community responses in two MSL systems with different clay contents (8% and 54%) exposed to MC-contaminated inputs (well water and eutrophied lake water). Samples were analysed before and after treatment using quantitative PCR (qPCR) to quantify the mlrA gene (encoding microcystinase) and its bacterial hosts. Next-generation sequencing (NGS) was used to assess microbial diversity, while the FAPROTAX database was used to predict functional characteristics. Results showed that MC was mainly adsorbed in pozzolan layers, while mlrA gene abundance and MC-degrading bacteria were higher in soil mixture layers. The presence of mlrA and associated bacteria was most pronounced in lake inflow samples, indicating intrinsic MC Biodegradation potential. Taxonomic analysis revealed dominant phyla including Proteobacteria, Actinobacteriota, Firmicutes, Chloroflexi and Bacteroidota. Functional analysis identified dominant traits such as chemoheterotrophy and aerobic metabolism. These findings provide new insights into microbial interactions in MSL systems and contribute to the optimisation of water treatment strategies for MC-contaminated environments.