Tunisia is located at a key position in the Mediterranean Sea where it constitutes a transitional area between the eastern and western basins benefitting from rich habitat diversity. The most recent inventory of marine macrophytes dated back to 1987 with an update in 1995, however these inventories were performed with identification based on morphological observations, which can be misleading for many seaweed species. To the best of our knowledge, this is the first study of the Tunisian macroalgal flora using DNA barcoding methods. Our target was to carry out a survey of macroalgae, to reveal cryptic species and allochthonous introductions, and to identify taxa with problematic taxonomy. COI-5P sequence analysis resolved 26 genetic species, including two new reports and three alien species. Of the remaining taxa, several displayed cryptic diversity relative to species reported from the Mediterranean versus from other geographical regions with taxonomic and nomenclature updates provided where possible. The aim of the present paper was to provide a DNA barcode-assisted floristic list of florideophycean algae collected along the Tunisian coast.