Background: This study aimed to investigate the impact of in vitro rumen fermentation (IVRF) on the microbiome structure and composition of rumen fluid before and after fermentation assays. Methods and Results: Six separate fermentation batches were run for 48 h using maize silage as the basal feed. Rumen fluid samples were analyzed before (RF; only rumen fluid inoculant) and after 48 h fermentation assay (MS; maize silage as the substrate) and further processed for microbiome analysis using amplicon sequencing targeting the V4 region of the bacterial 16S rRNA gene. Bacterial alpha diversity revealed that the Shannon index and observed index were similar between MS and RF fluid. The core microbiome was detected in 88.6% of the amplicon sequence variants in MS and RF. Taxonomic analysis at the phylum level showed similar abundances of Bacteroidetes, Proteobacteria, Firmicutes, Verrucomicrobiota, Spirochaetota, Patescibacteria, and Campilobacterota in MS and RF. The Bray–Curtis distance matrix showed similar bacterial community structure among MS and RF samples. Conclusion: Our results indicated that the in vitro procedure did not affect the bacterial community structure compared to the original rumen fluid inoculum. It should be noted that assessing the microbiome at a single endpoint (i.e., 48 h) may not provide a comprehensive understanding of the microbiome profile dynamics. However, the findings of this study provide a basis for future microbiome-based in vitro fermentation tests and confirm that the technique allows a high degree of species diversity that approximates the rumen function in vivo.