Annually, the wine industry produces high amounts of waste that can be toxic if disposed of without pretreatment. Vermicomposting is an efficient and low-cost method of decomposing organic matter using earthworms under controlled conditions. The organic substrate used in the vermicomposting process affects microbial populations and reflects the dynamics of enzymatic activity, decomposition of organic matter, and nitrogen transformations. However, the microbiome associations during the whole process are still unexplored. Thus, the aim of the present study was to investigate physicochemical, enzymatic, microbial, and microbiome activities during vermicomposting of winery waste. In this vein, a rectangular vermireactor with Eisenia andrei and Eisenia fetida earthworms, loaded with winery waste, was used. At the end of the process, the carbon/nitrogen (C/N) ratio was decreased, the total nitrogen was increased, the pH was neutralized and excess enzymatic activities were recorded. The bacterial and fungal phyla detected by next-generation sequencing analysis identified Armatimonadetes, Bacteriodetes, Candidatus saccharibacteria, Chloroflexi, Cyanobacteria, Planctomycetes, and Proteobacteria and Ascomycota, Basidiomycota, Chytridiomycota, Entomophthoromycota, Glomeromycota, and Mucoromycota, respectively. Physicochemical and microbial changes occurring during vermicomposting of winery waste, along with the microbiome diversity at the beginning and end of the process, may lead to a better understanding of winery-waste biotransformation into effective biofertilizer.