Ruminal ciliates are linked to methane production and nitrogen utilization efficiency in ruminants due to their association with other ruminal microorganisms. However, research on the specific interplay between ruminal bacteria and ciliates is still limited, particularly in different dietary conditions. This study examines the effect of the forage-to-concentrate (F:C) ratio on the ruminal bacteriome in vitro, focusing on bacteria associated with Isotricha spp. and small entodinia. The rumen fluid used as the inoculum for this experiment was collected from two cannulated Hanwoo cows. Dietary treatments included high-forage (HF, F:C of 7:3), high-concentrate (HC, F:C of 3:7), and control (CON, F:C of 5:5). After 24-hour incubation, fractions for entodinia-associated bacteria (EAB), Isotricha-associated bacteria (IAB), and total bacteria (TB) were collected for bacteriome analysis using QIIME2 with full-length 16S rRNA gene sequences on the PacBio system. All fermentation parameters, except for NH-N, showed linear changes with increasing F:C ratios (p ≤ 0.05). F:C ratio affected Isotricha spp. and Dasytricha spp. counts. Ciliate-associated bacterial fractions were significantly less diverse than the total bacterial group, as indicated by richness, phylogenetic diversity, and evenness indices. This suggests potential specific associations within ciliate-provided microhabitats. Both diet and ciliate fractions significantly influenced the overall bacteriome (p ≤ 0.05). More bacteriome features were differentially abundant due to the ciliate fraction effect rather than diet (q ≤ 0.05). Our newly proposed washing procedure, using higher ciliate cell counts and minimal bacterial contamination, effectively removed free-living or loosely associated bacteria. This allows focus on ciliate-associated bacterial populations, which may include potential symbionts or engulfed bacteria of host ruminal ciliates. Verifying these associations could provide insights into rumen microbiome dynamics, nitrogen utilization, hydrogen balance, and microbiome variation under different F:C ratios.