Background and objectives: Biochar is a carbonaceous material obtained when burning agriculture and animal biomass in a closed container with little or no available oxygen. Biochar combines a porous structure and large surface area that create habitat for establishment and development of biofilm residing microbes in anaerobic environments including rumen and increased microbial growth, reproduction and improved efficiency of fermentative digestion. Highly porous structure of biochar such as methanotrophes (methane oxidants) facilitates the formation of microbial colonies. Methane oxidation, archae and methanotrophic consortia on to the surfaces of the biochar increase as well as their population. The objective of this study was to determine effect of different levels walnut shell and chicken manure biochar on gas production, digestibility, methane, and ammonia production in an in vitro batch fermentation system. Materials and methods: Sun-dried walnut shell and chicken manure heated in a closed container with little available oxygen. Levels of 0. 5, 1 and 1. 5% of each biochar were added to the experimental diet including alfalfa, wheat straw, barley grain, corn grain, soybean meal, vitamin and mineral supplement and salt with ratio 60% forage to 40% concentrate. The study was carried out in a completely randomized design with seven treatments: 1-Control, without biochar; 2-basal diet+ 0. 5% walnut shell biochar; 3-basal diet+ 1% walnut shell biochar; 4-basal diet+ 1. 5% walnut shell biochar; 5-basal diet+ 0. 5% chicken manure biochar; 6-basal daiet+ 1% chicken manure biochar; and 7-basal diet+ 1. 5% chicken manure biochar. All samples were incubated with three replications using buffered rumen fluid obtained from four Kermanian sheep (two years old and 50. 5 kg weight) for 2, 4, 6, 8, 12, 18, 24, 48, 72 and 96 hrs. At the end of 24 hrs of incubation gas production, dry matter digestibility, concentrations of ammonia and methane was measured and gas production parameters, metabolizable energy, short chain fatty acids and organic matter digestibility were estimated. Results: The result showed that the gas production decreased (P<0. 01) by addition of biochar to diets after 24 and 96 hrs of incubation, but the level of 1% of walnut shell biochar and 1. 5% of chicken manure biochar had minimal impact on gas production. Means of metabolizable energy, short chain fatty acids and digestibility of organic matter at the level 1% of walnut shell biochar and 1. 5% chicken manure biochar were not different from control. Digestibility of dry matter in vitro increased at the 1% level of walnut shell biochar (p<0. 01) and 1. 5% of chicken manure biochar (p<0. 01). Addition of 1% walnut shell biochar and 1. 5% chicken manure biochar decreased ammonia-N concentrations, methane and methane production compared to control. Conclusion: In general, these findings indicated that the 1% walnut shell biochar and 1. 5% chicken manure had a potential to decrease methane mitigation and ammonia concentrations. Therefore, they can be new alternatives for modifying of rumen fermentation.