A novel method was developed for virus-like particle (VLP) extraction and characterization from biological soil crust (BSC) after microbial community reactivation. The method consisted of a single cell analysis by flow cytometry to monitor viable cells in BSC reactivated under controlled hydration, temperature, and light/dark exposure. Then, VLPs were extracted from reactivated BSCs, followed by viral DNA extraction and shotgun metagenomic analysis. The hydrated BSC under light/dark conditions showed the highest number of viable cells, and this condition was optimal for VLPs isolation. Taxonomic composition showed that families of the order of Caudovirales (Podoviridae, Myoviridae and Syphoviridiae) were the most abundant double strand DNA phages while Microviridiae were the most abundant single strand DNA phages. The isolated VLPs also carried sequences of relevant bacterial inhabiting soil. The functional categories of “phages, prophages, transposable elements, plasmids” and “clustering-base subsystem” were abundant (38 and 12%, respectively). All these data suggest viral predation as a key factor in shaping and maintaining bacterial diversity in the BSCs.