Madagascar is a marine biodiversity hotspot. A recent checklist recorded 1689 marine or transitional water fish species, 2.5% being endemic. To date, studies in this country were mostly focused on adult fishes using morphological-based identification. The early life stages of fishes remain largely understudied. The present work aimed to improve knowledge of fish biodiversity in Madagascar by focusing on post-larval reef fishes and settled juveniles in seagrass meadows of southwest Madagascar by using either species identification keys or DNA barcoding. Up to 119,500 individuals were collected, and 1096 individuals were successfully barcoded. We identified 387 species—85 through their morphology (with 58 unsuccessfully sequenced) and 302 by using CO1 barcoding corresponding to 302 barcode index numbers (BINs). This study added 27 new BINs for the BOLD database, 120 new for Madagascar, but only 159 were assigned a precise species name. By referring to the updated checklist of Madagascar fishes, 10 new species were detected for Madagascar. This number will probably increase when all the barcoded specimens become assigned to precise species names. These preliminary findings stress our poor knowledge of marine fish biodiversity in Madagascar and demonstrate the relevance of DNA barcoding in improving this knowledge.