Glyphosate-based herbicides are among the most used non-selective herbicides worldwide and inhibit synthesis of aromatic amino acids in plants, bacteria, and fungi. Given the broad usage, controversies concerning potential effects of glyphosate on health and especially on gut microbiomes arose. For cattle, it has been proposed based on in vitro data that glyphosate has detrimental effects on the ruminal microbiome, which manifest as a specific inhibition of bacteria involved in fiber degradation and as an enrichment of specific pathogens. In the present study, glyphosate effects on the ruminal microbiome were analyzed in vivo using glyphosate contaminated feedstuffs with strong differences in dietary fiber and dietary energy content in order to reproduce the proposed detrimental glyphosate effects on the rumen microbiome. While significant impact of dietary factors on the ruminal microbiome and its products are pointed out, no adverse glyphosate effects on ruminal microbiome composition, diversity, and microbial metabolites are observed.