The microbial diversity of a Tunisian olive fermentation brine was analysed using a culture-independent approach based on the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). SSCP patterns show a remarkably simple microbial community but higher for bacterial community than for the eukaryotic community. This study did not show the presence of archaeal populations. After PCR amplification, two small subunit (SSU) rRNA clone libraries of Bacteria and Eucarya populations were established. Three bacteria and only one eukaryotic phylotype were identified. Two dominant bacteria showed 100% phylogenetic similarity to the 16S rRNA sequences of Lactobacillus plantarum and Lactobacillus collinoides and represent 85% of bacterial community. The third bacteria phylotype was phylogenetically close related to the 16S rRNA sequence of a moderately halophilic bacterium belonging to the class gamma Proteobacteria. The dominant eukaryotic phylotype was identified as a Pichia membranaefaciens.