Turkey has a great number of karstic caves which are unexplored and have unknown microbial diversity. The biodiversity characterisation of these caves has not yet been systematically studied from the molecular point of view. Çal Cave in Trabzon, Turkey is one of the important karstic caves. In the present study, a metagenomic approach was used to explore the microbial diversity of Çal Cave for the first time to assess the potential of gene sources. Detailed taxonomic profiling was defined by sequencing all environmental genomes instead of a specific marker gene such as 16S rRNA which only targets prokaryotes. Taxonomic analysis revealed that the Çal Cave soil sample was represented as 98% Bacteria, 2% Eukaryota, 0.3% Archaea, and 0.01% Virus. Results showed that, the 31 distinct bacterial phyla represented in the Çal Cave soil sample were dominated by Actinobacteria (65%) and Proteobacteria (31%). The most dominant bacterial genus was Streptomyces. Among the 2% Eukaryotic population, the largest phylum was Ascomycota and it was mostly represented as Sordariomycetes. It was determined that 77% of Archaea was Halobacteria. The most abundant class of viruses dwelling in Çal Cave was Caudovirales. 91.61% of total readings could not be classified into any specific kingdom. Overall, classified and unclassified data verify that there exists vast microbial biodiversity in Çal Cave which could not be identified with classical microbiology techniques, and this microbial diversity provides a promising gene source for novel enzyme and bioactive compounds to be used in biotechnological applications.