Bacterioplankton play a central role in energy and matter fluxes at the sea, yet the factors that constrain their variation in the marine systems are still poorly understood. Here we show the explanatory power of multivariate statistical analysis of bacterial community structures coupled with fine measurements of numerous environmental parameters. We gathered and analysed data from a one month sampling period from the surface to 1000 m depth at the JGOFS-DYFAMED station (NW Mediterranean Sea). This station is characterized by very poor horizontal advection currents what makes it an ideal model to test hypothesis on the causes of vertical stratification of bacterial communities. Capillary electrophoresis single strand conformation polymorphism (CE-SSCP) fingerprinting profiles analyzed using multivariate statistical methods demonstrated a vertical zonation of bacterial assemblages in three layers, above, in or just below the chlorophyll maximum and deeper, that remained stable during the entire sampling period. Through the use of direct gradient multivariate ordination analyses we demonstrate that a complex array of biogeochemical parameters is the driving force behinds bacterial community structure shifts in the water column. Physico-chemical parameters such as phosphate, nitrate, salinity and to a lesser extend temperature, oxygen, dissolve organic carbon and photosynthetically active radiation acted in synergy to explain bacterial assemblages changes with depth. Analysis of lipid biomarkers of the organic matter sources and fates suggested that bacterial community structure at the surface layers was in part explained by lipids from chloroplast origin. Further detailed analysis of pigment-based phytoplankton diversity gave evidence of a compartmentalized influence of several phytoplankton groups on bacterial community structure in the first 150 m depth. This study is probably the first example of an analysis employing a complex environmental dataset in combination with microbial community profiles to unravel the mechanisms underneath bacterial assemblages in marine systems.