This study examined the potential of duckweed powder (DWP) on in vitro fermentation characteristics, nutrient degradability, microbial change, and methane (CH
4
) production using in vitrostudy technique. This investigation used a 2 × 6 factorial arrangement in a completely randomized design (CRD) by different roughage-to-concentrate (R: C) ratios of 60:40 and 40:60 combined with DWP supplementation levels at 0, 2, 4, 6, 8, and 10% of the total dry matter (DM) substrate, respectively. There was an interaction effect by R: C ratios combined with DWP supplementations that changed gas production rate, pH value (4 h; h), volatile fatty acid (VFA) (8 h), in vitro dry matter degradability (IVDMD) at 12 h, and concentration of ammonium nitrogen (NH
3
-N) (
p
< 0.05). Furthermore, the R: C ratio (40:60) significantly decreased CH
4
production (4 and 8 h), pH (8 h), and
Ruminococcus albus
(8 h) (
p
< 0.05), while it significantly increased total VFA (8 h), and nutrient degradability (
p
< 0.05). DWP 4% significantly increased to the highest of gas production, improved nutrient degradability (IVDMD at 24 h and in vitro organic matter degradability; IVOMD at 12 h), whereas significantly decreased
Methanobacteriales
(8 h) and CH
4
production. DWP 4% has potential as a ruminant feed additive for reducing
Methanobacteriales
and CH
4
emission and enhancing rumen fermentation.