Marine free-living (FL) and plankton-associated prokaryotes (plankton-microbiota) are at the basis of trophic webs and play a crucial role in the transfer and cycling of nutrients, organic matter, and contaminants. The local environmental conditions differ drastically according to the plankton type and size fraction due to different metabolisms of pico-, nano-, micro-phytoplankton and zooplankton. Despite its relevant ecological role, the plankton-microbiota has been rarely investigated with a sufficient level of size-fraction resolution, and can be challenging to study because of overwhelming amounts of eukaryotic host DNA. Here we compared the prokaryotic diversity obtained by 16S rRNA gene sequencing from six plankton size fractions (from FL to mesoplankton), through three DNA recovery methods: direct extraction, desorption pretreatment, enrichment post-treatment. The plankton microbiome differed strongly according to the plankton size-fraction and methodology, and we identified prokaryotic determinants of each size fraction and methodology.