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Abstract Angkor temples in Cambodia, an icon of Khmer civilization, display the ancient culture by bas-relief on the sandstone surface of different temples, which are being destroyed by physical, chemical and biological processes for more than a thousand years. To investigate the bio-erosion of temple sandstone at the Royal Palace of Angkor Thom and Beng Mealea in Cambodia, Next Generation Sequencing (NGS) Illumina sequencing technology based on 16S rRNA gene was performed on samples of biofilm and exfoliated sandstone materials to identify the microbial community composition. After quality filtering the raw data, 678,115 quality reads were obtained for bacterial 16S rRNA gene from a total of 13 samples with high Goods coverage and satisfactory rarefaction curves. Higher bacterial diversity was detected in exfoliated sandstone materials than the biofilms, but the lowest in the lower layers of the biofilm than the top layers. At the phylum level, 4 phyla, namely Proteobacteria, Actinobacteria, Acidobacteria, and Chloroflexi, were the most common and dominant bacterial groups in these samples with each contributing to greater than 3.7% of the total abundance. Both Firmicutes and Gemmatimonadetes were the dominant phyla detected only in exfoliated materials, while Cyanobacteria, Chloroflexi, and unassigned bacteria were more abundant in the biofilms. Hierarchical cluster analysis at the genus level showed that the distribution of bacterial community composition between exfoliated materials and biofilms was significantly different. The microbiota of Beng Mealea and the Royal Palace was different, especially for the biofilm samples. The correlation of environmental factors and bacterial community structure suggested that the nitrification process was more active at Ben Mealea, which might contribute to biodeterioration. This analysis of microbiota in these biofilms and sandstone exfoliation materials provides further information on the responsible microorganisms involved in geobiochemical processes at Angkor monuments and preservation strategies under tropical climate conditions. Graphical abstract Image 1 Highlights • NGS Illumina sequencing of biofilm and exfoliated sandstone materials at Ben Mealea and the Royal Palace. • Microbiota of the Beng Mealea and the Royal Palace was different, especially for the biofilm samples. • Higher bacterial diversity in exfoliated sandstone than biofilms. • Lowest diversity in the bottom layer of the biofilm comparing with the upper layers. • Nitrification process was more active at Ben Mealea than the Royal Palace. [ABSTRACT FROM AUTHOR]