Highly stratified karstic estuaries (salt-wedge estuaries) are characterized by a strong gradient of salinity and other environmental parameters. The bacterial diversity in many estuaries has been documented but no information is available about the diversity of bacteria in Mediterranean karstic estuaries. A combination of 454 pyrosequencing of the 16S rRNA gene and catalyzed reporter deposition-fluorescence in situ hybridization techniques were used to estimate the bacterial diversity across the karstic Krka River Estuary in February and July 2013. The bacterial community at the OTU level showed stronger seasonal, rather than spatial, variation that is caused by the phytoplankton bloom in February. The alphaproteobacterial SAR11 clade was an important group whose abundance showed a decrease toward the low salinity riverine water. Higher relative abundance of the SAR11 clade was detected in July when more oligotrophic conditions were observed. Roseobacter responded to the phytoplankton bloom in February increasing its abundance in the water layer with higher phytoplankton biomass. Cyanobacteria showed substantial seasonal change being an important primary producer in July during lower nutrient conditions. Actinobacteria and Betaproteobacteria were characteristic for the freshwaters layer of the estuary with Actinobacteria showing strong seasonal change and higher abundance in July. Bacteroidetes and Gammaproteobacteria also showed seasonal differences with higher abundances in February during the phytoplankton bloom in the inner part of the estuary. The bacterial community in the Krka River Estuary showed pronounced seasonal changes, due to the phytoplankton bloom observed in February, and vertical changes, as a result of a stable, sharp halocline that is characterizing this type of estuaries.