In Tunisia, several food products derived from meat or seafood are naturally processed, without any addition of bacterial starters. Such fermented, dried-cured, salted, or marinated products, as well as the raw meat or fish may thus provide a source to isolate the natural microflora colonizing such environments. We isolated lactic acid bacteria from a representative range of flesh-foods sold or manufactured in different parts of Tunisia, and selectively searched for Lactobacillus sakei, a lactic acid bacterium potentially useful as starter or protective culture. Eighty six (86) strains were isolated from various seafood (anchovy, sardine, sole, mullet, and octopus), or meat (pork, veal, beef, sheep, chicken, and turkey) products that were either fresh, or transformed by different traditional processes. Several methods were used in order to develop a rapid and reliable protocol for the direct identification of L. sakei. Amplified ribosomal DNA restriction analysis (ARDRA) classified the various isolates into 9 distinct groups. Search for the presence of the L. sakei specific katA gene indicated that all positive isolates were grouped in the same ARDRA group. Sequencing of 16S rDNA confirmed those isolates as L. sakei. Those 22 different L. sakei strains represent 25.6% of the total isolates, while other isolates found in the different ARDRA groups were tentatively ascribed to Lactobacillus plantarum, Lactococcus lactis/garviae, Enterococcus avium, Streptococcus parauberis, Hafnia alvei, Pediococcus pentosaceus, and Lactobacillus curvatus through 16S rDNA sequencing. A fast and reliable method to isolate and discriminate L. sakei from complex food environments is proposed.