Biochar is a novel carbonized feed additive sourced from pyrolyzed biomass. This compound is known to adsorb gasses and carbon, participate in biological redox reactions and provide habitat biofilms for desirable microbiota proliferation. Therefore, biochar holds potential to modify rumen fermentation characteristics and reduce enteric CH
4
emissions. The objective of this study was to investigate the effect of hardwood biochar supplementation on fermentation parameters, methane (CH
4
) production and the ruminal archaeal, bacterial, and fungal microbiota using the
in vitro
RUSITEC (rumen simulation technique) system. Treatments consisted of a control diet (oaten pasture: maize silage: concentrate, 35:35:30 w/w) and hardwood biochar included at 400 or 800 mg per day (3.6 and 7.2% of substrate DM, respectively), over a 15-day period. Biochar supplementation had no effect (
P
≥ 0.37) on pH, effluent (mL/d), total gas (mL/d), dry matter (DM) digestibility or CH
4
production (mg/d). The addition of 800 mg biochar per day had the tendency (
P
= 0.10) to lower the % of CH
4
released in fermentation compared to 400 mg/d biochar treatment. However, no effect (
P
≥ 0.44) was seen on total VFA, acetate, propionate, butyric, branched-chain VFA, valerate and caproate production and the ratio of acetate to propionate. No effect (
P
> 0.05) was observed on bacterial, archaeal or fungal community structure. However, biochar supplementation at 800 mg/d decreased the abundance of one Methanomethylophilaceae OTU (19.8-fold,
P
= 0.046) and one
Lactobacillus
spp. OTU (31.7-fold,
P
< 0.01), in comparison to control treatments. Two fungal OTUs classified as
Vishniacozyma victoriae
(5.4 × 10
7
increase) and
Sporobolomyces ruberrimus
(5.4 × 10
7
-fold increase) were more abundant in the 800 mg/d biochar samples. In conclusion, hardwood biochar had no effects on ruminal fermentation characteristics and may potentially lower the concentration of enteric CH
4
when included at higher dosages by manipulating ruminal microbiota abundances.