The aim of the study was to investigate microbial succession in the mushroom supply chain from compost, casing to fruit body formation and mushroom growth to the point of harvested, packing and point of sale. The microbial population dynamics of compost, casing and mushrooms were determined using a plate count technique, denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S and 18S rDNA. Plating revealed greater abundance of bacteria, fungi and yeasts in mushroom compost compared to casing and fresh mushroom samples. The viable count method also showed that bacteria and yeasts increased significantly after harvest and Page 2 of 38 during cold storage. Sequencing revealed a more diverse culturable bacterial population in casing and on the mushrooms than in the compost. Phylogenetic analysis revealed a general trend of grouping of species from the same sources. In contrast, a higher microbial diversity was recorded in compost when using the DGGE method, which reflects cultural and non-culturable microorganisms. For compost and casing bacteria studied using DGGE, several species formed separate lineages, demonstrating highly diverse communities in these samples. Fungi were shown to be less abundant and less diverse compared to bacteria and yeasts. The study provides baseline knowledge of microbial populations and -succession trends in mushroom production systems using viable and non-viable methods. The information provided in this study may be useful for microbial ecology studies and to identify and develop biocontrol systems for pathogen control during production or to enhance pinning stimulation by knowing when to apply Pseudomonas spp. to ensure increased yield. Finally an insight is provided into microbial survival during cold storage and marketing of mushrooms. Potential antagonistic populations known to prevent spoilage, quality deterioration and extend shelf life are listed in this paper.