Supplementation of appropriate probiotics can improve the health and productivity of ruminants while mitigating environmental methane production. Hence, this study was conducted to determine the effects of
Enterococcus faecium
SROD on in vitro rumen fermentation, methane concentration, and microbial population structure. Ruminal samples were collected from ruminally cannulated Holstein–Friesian cattle, and 40:60 rice straw to concentrate ratio was used as substrate. Fresh culture of
E. faecium
SROD at different inclusion rates (0, 0.1%, 0.5%, and 1.0%) were investigated using in vitro rumen fermentation system. Addition of
E. faecium
SROD had a significant effect on total gas production with the greatest effect observed with 0.1% supplementation; however, there was no significant influence on pH. Supplementation of 0.1%
E. faecium
SROD resulted in the highest propionate (
P
= 0.005) but the lowest methane concentration (
P
= 0.001). In addition, acetate, butyrate, and total VFA concentrations in treatments were comparatively higher than control. Bioinformatics analysis revealed the predominance of the bacterial phyla
Bacteroidetes
and
Firmicutes
and the archaeal phylum
Euryarchaeota
. At the genus level,
Prevotella
(15–17%) and
Methanobrevibacter
(96%) dominated the bacterial and archaeal communities of the in vitro rumen fermenta, respectively. Supplementation of 0.1%
E. faecium
SROD resulted in the highest quantities of total bacteria and
Ruminococcus flavefaciens
, whereas 1.0%
E. faecium
SROD resulted in the highest contents of total fungi and
Fibrobacter succinogenes.
Overall, supplementation of 0.1%
E. faecium
SROD significantly increased the propionate and total volatile fatty acids concentrations but decreased the methane concentration while changing the microbial community abundance and composition.