With the advent of contemporary molecular tools, the conventional microbiological isolation, enrichment techniques, and approaches have changed considerably. Molecular techniques such as polymerase chain reaction, cloning, and sequencing have shown that the major percentage of microbial diversity in an ecosystem remain “unculturable” or “as yet uncultivable” due to the lack of information on their biology, limited selection media, and culture conditions that could support their growth. Identifying and knowing more about them have become an important objective in the microbiological research. The ecological, environmental, and functional implications of a microbial ecosystem can be deciphered by knowing its microbial composition and interactions. The areas of whole-cell and targeted gene metagenomics are playing a key role in accomplishing this objective. The present review discusses the 16S ribosomal RNA (16S rRNA) gene metagenomics approach, which has found major applications in identifying the composition of a given microbial ecosystem. Different systems, processes, and analysis tools are available to perform 16S rRNA metagenomics; however, there are few concerns that require more investigation to gain the maximum benefit of these techniques.