Biogas plants are one of the most stable sources of renewable energy. Currently, there is a noticeable increase in the amount of post-production residues from agricultural production and agri-food processing (fruit and vegetable processing, fermentation, beet pulp, or lignocellulosic waste), which, can be used for biogas production after appropriate pretreatment. The aim of this study was to examine the possibility of using the biomass produced during the cultivation of grapes on a selected farm as a substrate for a biogas plant, taking into account the production process. The research was carried out in 2018–2020 in a vineyard located in the Sandomierz Upland in the south-eastern part of Poland. Own rooted vines were grown as a single continuous string with a trunk height of 40 cm and a length of one fixed arm approx. 0.9 m, on which six pivots were left every year after applying a short cut, from which 12–16 fruit shoots were derived, the so-called grapevines. Leaves were collected at random from three locations on the fruiting shoot, a total of 30 leaves in each replicate. Each sample consisted of 1/3 of the leaves collected at the bottom, 1/3 in the middle, and 1/3 at the top of the canopy. Leaf area was estimated with a model 3100 area meter on a sample of 30 leaves from each replicate. Both the quantity and quality of the obtained material as a substrate for methane fermentation were evaluated. Biogas yield tests in optimal conditions for mesophilic bacteria were conducted on three substrate samples referred to as ‘Regent’, ‘Seyval Blanc’, and ‘Solaris’. The yields of the tested material ranged from 51.0 to 59.0 Nm3 biogas per Mg of biomass.