Within Australia, approximately 6.4% of total greenhouse gas emissions are from animal methane (CH4) derived from enteric fermentation. Mitigation of ruminant CH4 is a key concept in support of sustainable agriculture production; dietary manipulations a viable strategy to lower CH4 release during enteric fermentation. In order to determine the effects of dose response of biochar and wood vinegar supplementation on fermentation parameters and CH4 production, this study utilized in vitro batch culture incubations. It is hypothesized that the addition of either biochar or wood vinegar will successfully reduce enteric CH4 emissions without negative modification of other fermentation parameters. Three feed substrates (vegetable mixed ration, maize silage, and winter pasture) were separated into treatments containing either biochar at 0%, 0.5%, 1%, 2%, and 4% DM replacing substrate (w/w basis), or wood vinegar at 0%, 0.25%, 0.5%, 1%, and 2% into incubation media volume (v/v). At 6, 12, and 24 hours after inoculation, total gas volume, and methane (CH4 %) were measured. Volatile fatty acid (VFA) concentrations, media pH, and in vitro dry matter digestibility were measured at 24 hours. Biochar at various dosages had no effect (P > 0.05) on fermentation characteristics other than decreased in vitro dry matter digestibility (IVDMD; P = 0.01) at 2% and 4% (DM basis) inclusion. Similar to biochar, dose response of wood vinegar had no effect on in vitro fermentation characteristics. However, feed substrate had major effects on all fermentation parameters (P = 0.01) where winter pasture > vegetable mixed ration > maize silage for all recorded fermentation characteristics. Biochar and wood vinegar supplementation were ineffectual in mitigating CH4 production or modifying fermentation characteristics, thus rejecting the initial hypothesis. These results suggest the use of biochar is not an effective tool for methane mitigation in ruminant livestock and infers that studies previously reporting success must better define the systemic mechanisms responsible for the reduction in CH4.