Tunisia has limited primary energy resources and produces only fossil fuels. The technology of biogas production by anaerobic digestion or biomethanation is very little known and poorly applied in Tunisia. Indeed biogas is very neglected among the main sources of energy in the country. The use of biogas technology can solve a number of ecological and economic problems. Today, its prospects for the future appear promising, justifying the need for a judicious awareness, followed by pilot experiments with a view to the further popularization of biogas technology and its interests. In Tunisia, the poultry sector produces a large quantity of laying inches, abandoned without any recycling or recovery, which can constitute a potential source of an environmental contamination in general, although it is an organic source that can be valued and has important energy and agronomic interests. This study aims to optimize and to follow the experimental biometanation of the poultry litter. Physicochemical and microbiological evaluation of the substrates was carried out. As for the energy side, the anaerobic digestion chosen is in discontinuous (batch), mesophile (35 ° C) and wet (8% DM of digester) mode. The main results show that poultry excreta is rich in organic matter and bacterial load, which favors the production of biogas, in particular with the addition of inoculum and activator. It should also be noted that anaerobic digestion resulted in a decrease in microorganisms and that the activator (especially with 18%) improved the speed of the hydrolysis phase and favored the production of biogas and especially methane.