In biogas plants, almost any type of organic matter can be used as a substrate to produce biogas. To make the process of methane fermentation more effective, these materials are pretreated. This applies in particular to a group of difficult substrates. Straw, due to its hemicellulose structure and saturation, is hardly fermented by biogas reactor microorganisms. The methods of post-harvest residue preparation for anaerobic digestion being applied so far are expensive, while their application has a negative effect on methanoegenic bacteria. Therefore, the microorganisms being able to degrade straw hemicellulose structure, utilisation of which could precede the proper fermentation process, have been searched for. This paper presents the results of microbial biodiversity analysis in the environmental samples being lupin, cereal, rape and maize straw as well as hay and haylage at different degradation stages. The analysis of biodiversity will help at a further stage of study to isolate active microbial strains showing cellulolytic, hemicellulolytic or ligninolytic activity which are desirable in the process of straw biodegradation. Analysis of the microbial count was performed by the method of deep inoculation on different microbiological culture media. The conducted tests include determination of the number of fungi, bacteria and actinomycetes. The results obtained confirm the usefulness of the analysed samples for isolation of microbial strains capable of converting straw preceding the biogas production.