Abstract. Bacterial population distribution in the Mediterranean Sea has been mainly studied by considering small geographical areas or specific phylogenetic groups. The present study is a molecular microbiology investigation aimed to identify the environmental factors driving total bacterioplankton community composition of seawater samples collected along a transect covering the whole Mediterranean Sea. We performed automated ribosomal intergenic sequence analysis (ARISA) and microscope evaluation of prokaryotic abundance of seawater sampled across both vertical profiles and longitudinal transects in the whole basin. Prokaryotic abundance decreased with depth at all the stations and presented similar values in epi-, meso- and bathypelagic layers across the whole Mediterranean Sea. However, peculiar bacterial assemblages were selected along a longitudinal transect in the epipelagic layers of the eastern and western sub-basins. Vertical differences of the bacterial communities were observed only when considering the epi- and bathypelagic waters, while the study of the structure of bacterial communities at a finer scale across the water column displayed higher variability at the intermediate layers. Nonetheless, different physico-chemical factors were significantly related to the distribution of bacterial populations, both according to geographic position and down the water column in the whole Mediterranean Sea. These results demonstrated that bacterial assemblages are putatively correlated to different water masses of the complex hydrographical systems of the eastern and western Mediterranean sub-basins.