Historically used in textile and paper industry, hemp fibres have started to find new applications in composite materials with important economic and ecological advantages. However, their applications are limited since manufacturers have some difficulties to standardise fabrication processes. This study is a first step before selection and isolation of strains that could later be used to optimise microbial retting efficiency and hence fibre quality. We studied six samples harvested on different ground types, at different dates and with different retting durations on field to obtain an exhaustive representation of the process. After DNA extraction, total bacteria and fungi associated with stems during retting were specifically quantified using real-time PCR. Then, using sequence analysis of randomly cloned 16S and 18S ribosomal RNA (rRNA) genes, a phylogenetic characterisation of the dominant microorganisms was carried out. Quantitatively, we showed that there were 8.1-9.5 log₁₀ 16S rRNA gene copies per gram of hemp straw for bacteria and 8.6-9.6 log₁₀ 18S rRNA gene copies per gram for fungi. Qualitatively, we noticed a higher bacterial diversity in comparison to fungi. This work showed that in the different samples, the same species were present but in significantly different proportions according to ground type, harvest dates and retting durations on field. The most frequent bacterial sequences were affiliated to species Escherichia coli, Pantoea agglomerans, Pseudomonas rhizosphaerae, Rhodobacter sp., Pseudomonas fulva, Rhizobium huautlense and Massilia timonae, whereas fungal sequences were principally related to the genera Cladosporium and Cryptococcus.