Aims: Green-mould contamination is identified as one of the challenges faced by mushroom cultivation industry globally which believed to be caused by Trichoderma spp. Methods and Results:To explore the dynamics of microbial population in mushroom substrate during commercial mushroom cultivation and how microbiota might play a role in green-mould contamination, we applied both culturing and targeted metagenomics approaches to identify microbiota in noncomposted sawdust substrates at different cultivation stages. The microbiological analysis showed that the green-mould contaminated substrates harboured higher total mesophilic bacteria count. The green-moulds isolated from the contaminated mushroom substrates were identified as Trichoderma pleurotum (n = 15; 93.8%) and Graphium penicillioides (n = 1; 6.3%). To our surprise, the targeted metagenomic analysis revealed that Graphium comprised 56.3% while Trichoderma consisted of only 36.1% of the total fungi population, suggesting that green-mould contamination might not be caused by Trichoderma alone, but also Graphium that grows very slowly in the laboratory. Conclusion:It is worthwhile to note that G. penicillioides was also isolated in the early stages of mushroom cultivation, but not T. pleurotum. The results indicated that the structure and composition of the bacterial population in the mushroom substrate varied and the bacterial population shifted along the cultivation process. Significance and Impact of Study:This study revealed a possibility of G. penicillioides as an overlooked fungi causing green-mould contamination.