ABSTRACT Concerns over antibiotic resistance from the use of ionophore antibiotics in the treatment of metabolic disorders in cattle have led to investigations into alternative antimicrobial compounds. This is the first study to evaluate the effects of betulin, a natural compound found in birch tree bark, on in vitro rumen fermentation and its bacterial composition. Treatment with betulin decreased the relative abundance of Streptococcus and increased the relative abundance of Rikenellaceae RC9 gut group and Pseudobutyrivibrio without affecting short‐chain fatty acid (SCFA) production. Additionally, we found betulin had a minimum inhibitory concentration (MIC) of 18.75 μg/mL on Streptococcus equinus ATCC33317 and a MIC of 75 μg/mL on Pseudobutyrivibrio xylanivorans BAA‐455. Furthermore, we found a reduction in gas production in the betulin treatment group in batch culture and decreased viscosity of the culture medium incubated with S. equinus ATCC33317. These results suggest betulin's potential as a natural alternative to ionophore antibiotics to prevent metabolic disorders caused by Streptococcus .