In the search for natural alternatives to antibiotic feed additives, we compared the efficacy of two doses of Scrophularia striata extract [S. striata-Low at 40 and S. striata-High at 80 mg g-1 dry matter (DM)] with monensin (monensin) and a negative control in the modulation of rumen fermentation, methane production and microbial abundance in vitro. Microbes were investigated using qPCR and 16S rRNA targeted sequencing. Data showed that the addition of S. striata increased production of total short chain fatty acids (SCFA) in comparison to both monensin and control (P = 0.04). The addition of S. striata increased acetate production, and increased propionate at the higher dosage (P -0.71; P ≤ 0.001). The abundance of Fibrobacter genera and total SCFA (r = 0.86), acetate (r = 0.75), and valerate (r = -0.51; P