Abstract. Amorphous silica deposits found in orthoquartzite caves offer valuable analogues for understanding early life on Earth and potential biosignatures on Mars. This study presents the fully on-site microbial community analysis of silica stromatolite-like structures in the ancient and remote orthoquartzite cave Imawarí Yeutá (Auyan Tepui, Venezuela). Using a portable laboratory setup, we performed ATP-based microbial activity assessments and the full DNA-based analysis workflow directly in the cave, without internet access or high computational resources. The data obtained in the cave were then validated in the laboratory using a standard bioinformatics pipeline, qPCR and Biolog EcoPlate assays. The sequencing results revealed that the microbial communities in the stromatolite differ from other biofilms on the cave floor for the higher abundance of Actinobacteriota (particularly the genus Crossiella) and members of Subgroup 13 (Acidobacteriota) suggesting a possible role in the stromatolite formation/development. The ATP-based and Biolog results indicated that the most metabolically active microorganisms are localized in the white layer/colonies at basis of the stromatolite suggesting that the stromatolite development occurs at the interface of this structure with the quartz rock. These findings validate the feasibility of real-time microbial analyses in remote caves with astrobiological interest and provide novel understanding on the microbiological aspects involved in the formation of the silica stromatolites in non-thermal and aphotic environments.