From the time when microbial activity in wine fermentation was first demonstrated, the microbial ecology of the vineyard, grape, and wine has been extensively investigated using culture-based methods. However, the last 2 decades have been characterized by an important change in the approaches used for microbial examination, due to the introduction of DNA-based community fingerprinting methods such as DGGE, SSCP, T-RFLP, and ARISA. These approaches allowed for the exploration of microbial community structures without the need to cultivate, and have been extensively applied to decipher the microbial populations associated with the grapevine as well as the microbial dynamics throughout grape berry ripening and wine fermentation. These techniques are well-established for the rapid more sensitive profiling of microbial communities; however, they often do not provide direct taxonomic information and possess limited ability to detect the presence of rare taxa and taxa with low abundance. Consequently, the past 5 years have seen an upsurge in the application of high-throughput sequencing methods for the in-depth assessment of the grapevine and wine microbiome. Although a relatively new approach in wine sciences, these methods reveal a considerably greater diversity than previously reported, and identified several species that had not yet been reported. The aim of the current review is to highlight the contribution of high-throughput next generation sequencing and metagenomics approaches to vineyard microbial ecology especially unraveling the influence of vineyard management practices on microbial diversity.