Reuse of plant growing substrate can contribute to lowering the carbon footprint of horticulture production systems. Here, we assessed the impact on the root-associated microbiome of strawberries (Elsanta and Malling Centenary) grown hydroponically in fresh or up to three-times reused peat-based substrate, with or without steaming. Microbiome analysis (16S rRNA and ITS1 sequencing) revealed genotype-specific root microbiomes. Bacterial communities remained stable across reuse cycles, while fungal communities adapted more. Pathogenic fungi accumulated over time, highlighting the need for steaming to minimize infection risks.