This study used next-generation sequencing to assess the impact of combined capric acid (C10) and lauric acid (C12) on the ruminal bacterial composition. Eight Holstein cows were randomly assigned to two groups using a cross-over design. The cows were fed two silage-based diets with the addition of either 100 g of stearic acid per cow per day (control), or 50 g of capric acid and 50 g of lauric acid per cow per day (C10 + C12). On day 18, 250 mL of rumen fluid was collected from each cow, and DNA was isolated, amplified, and sequenced. Treatment did not alter bacterial diversity indices, the relative abundance of archaea, nor the fiber-degrading microorganisms, except for a decrease in Fibrobacter (from 2.9% to 0.7%; p = 0.04). The relative abundance of Prevotellaceae decreased (from 39.9% to 29.6%; p = 0.009), which is notable because some members help to efficiently utilize ammonia by releasing it slowly into the rumen. Furthermore, the relative abundance of Clostridia increased (from 28.4% to 41.5%; p = 0.008), which may have aided the increased ammonia-nitrogen levels in the rumen, as this class contains hyperammonia-producing members. Our study reveals alterations in bacterial abundances with implications for rumen ammonia levels, offering insights into potential strategies for modulating rumen fermentation processes and methane production in ruminant livestock.