Biochar, a carbon-rich product from the pyrolysis of biomass, has the potential to enhance feed efficiency and mitigate the environmental impact of milk production by optimizing rumen fermentation. However, its effect on dairy cow performance during peak production remains uncertain. We aimed to assess the potential of biochar to improve feed conversion efficiency and reduce ruminal methane production in high-yielding dairy cows. Additionally, through a combination of in vitro and in vivo experiments, we sought to distinguish between intra- and extra-ruminal effects of biochar. In the in vitro experiment, five levels of biochar (0%, 1%, 2%, 4%, and 8% of substrate DM) were incubated for 24 h in buffered rumen fluid with a substrate using a batch culture system. Biochar had minimal effects on in vitro fermentation, with no significant impact on key variables such as methane production or volatile fatty acid (VFA) concentrations. In the in vivo experiment, 34 lactating Holstein cows were used in a crossover design with two 29-day periods, each comprising a 20-day adaptation phase followed by a 9-day sampling phase. Two dietary treatments were: 1) Control, basal diet; 2) Biochar, basal diet supplemented with biochar (1% of DM). Biochar supplementation decreased milk yield (by 1.8 kg/d; P = 0.001), while dry matter intake (DMI) was not significantly affected (MINUS SIGN 0.4 kg/d; P = 0.080), resulting in lower feed efficiency. Additionally, biochar supplementation decreased yields of fat-corrected milk and energy-corrected milk. However, milk fat, protein, and lactose concentrations remained unaffected. Biochar increased rumen pH (6.43 vs. 6.71), acetate concentration, and the acetate:propionate ratio, while the effect on VFA and ammonia-N concentrations was not statistically significant. Cows fed biochar exhibited lower levels of blood urea nitrogen (BUN), Zn, and P, and showed no changes in blood vitamin levels. In conclusion, our findings suggest that high-yielding dairy cows do not benefit from biochar supplementation, as it decreases milk yield and feed efficiency. Our findings further indicate that the tested biochar is ineffective in reducing rumen methane.