The aim of this study was to determine the properties of the components used for fermentation and digestate. The content of phosphorus and its fraction in the substrate mixture undergoing fermentation in the real agricultural biogas plant and in the digestate was determined. The research was carried out based on substrate and digestate samples from an agricultural biogas plant with a production capacity of up to 1 MW (Mega Watt). The biogas plant operates in a continuous system. To ensure optimal operating conditions of the installation, it is necessary to provide substrates with appropriate quality and composition throughout the year. The substrate consists mainly of maize silage, poultry manure and potato pulp. In the study, samples of individual substrates and digestate were taken in four terms. The time of collection depended on the fraction of individual substrates in the charge composition. In the first term, the fraction of three substrates in the orchard was as follows: 79%—maize silage, 15%—poultry manure, and 6%—potato pulp. In the second term, it reached 82%, 10% and 8%, respectively, in the third 83%, 8% and 9%, and the fourth 80%, 6% and 14%. Eight samples of the substrates and digestate were collected every time. The samples were determined considering the content of dry matter, organic matter and the total content of phosphorus, magnesium, calcium, potassium, nitrogen, iron, and the pH value. After drying the samples, the fractions were determined by the phosphorus method with the Sequential Chang–Jackson extraction with subsequent modifications by Petersen and Corey. Measurements were made using the ICP-AES method. The following fractions were isolated: P-lab. (labile) labile phosphorus, P-Al phosphorus in aluminium phosphates, P-Fe phosphorus in gel phosphates, P-red.(reduced), P-ok. (occluded) the fraction of occluded phosphates absorbed on the surface of mineral particles, P-Ca phosphorus in calcium phosphates. It was found that the physicochemical composition of the feed and digestate was determined by the content of about 80% of maize silage in fermented substrates. The addition of substrates in the form of poultry manure and potato pulp influenced the content of total phosphorus and slightly modified the content of individual fractions in it. The fermentation process, to some extent, decreased the share of fraction I (mobile) and fraction II (combined with aluminium), increased the share of fraction VI (combination with calcium) and had no significant effect on the others. Digestate is a material rich in macronutrients and should be used as a fertilizer. The dosage of digestate used for fertilisation should be preceded by a knowledge of the soils and the requirements of the cultivated plants in order to prevent mobile phosphorus fractions from entering surface waters and increasing the eutrophication process.