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The mycotoxin-producing fungus Fusarium culmorum causes major feed spoilages in agricultural livestock, but effects of F. culmorum-contaminated feed on the structural diversity of the rumen-inhabiting microbial community are not understood. Avoiding animal experiments, this study was conducted with the rumen simulating technique (Rusitec). Small subunit (SSU) rRNA gene copy numbers of bacteria and archaea, determined by quantitative polymerase chain reaction (PCR), indicated no differences between contaminated and non-contaminated digested feed, but fungal copy numbers, not attributable to F. culmorum itself, were elevated approximately fourfold in the contaminated feed, with 2.3 × 109 g−1 dry weight. Single-strand conformation polymorphism profiles of PCR-amplified partial SSU rRNA genes revealed a single but clear difference between contaminated and non-contaminated feed in profiles encompassing the phylogenetic clusters of Fibrobacteriales and Clostridiales. Minor quantitative differences were also seen in profiles of archaea and fungi. Positive correlations were found between fungal rRNA gene copy numbers and the degradability of different nutrients, but there was no correlation with degradation rates of the major mycotoxin contaminant deoxynivalenol. Thus, the diversity responses of the microbial community to F. culmorum-contaminated feed were caused by a fungus-induced, altered feed quality rather than by direct mycotoxicity.