The environmental DNA (eDNA) technique characterizes spatio-temporal fish diversity patterns along estuarine habitats, such as hard-to-reach mangroves, yet it has rarely been applied in West Africa. In this study conducted over two seasons (dry and rainy) in coastal Guinea Bissau, fish eDNA was extracted from water samples collected in three habitats with distinct human uses. These habitats included site-specific mangroves, rice fields, and transition zones sampled in three sites: Elalab in Cacheu, Malafo in Oio, and Cafine in Tombali regions. As a reference, DNA barcoding of 181 fishes resulted in 243 DNA sequences for two mitochondrial markers (139 for COI and 104 for 12S), corresponding to 76 species (69 for COI and 56 for 12S), after morpho-genetic confirmation. The metabarcoding analysis detected 49 environmental DNA-Operational Taxonomic Units (eOTUs), attributed to 15 fish orders, 29 families, 38 genera, and 32 species. Fish abundance varied between sites and seasons but not between habitats. The southern Cafine site had a significantly lower abundance during the dry season, whereas the northern Elalab site hosted relatively fewer species during the rainy season. The transition from rice fields to mangrove habitat showed a positive trend in Shannon diversity with no fish being present in the rice fields during the dry season except for Malafo. Remarkably, Coptodon cf. guineensis, Sarotherodon melanotheron and Pseudotolithus elongatus dominated the fish assemblages. This pioneering eDNA study efficiently surveyed seasonal fish diversity within Bissau-Guinean mangroves, demonstrating its potential as a novel fish monitoring tool toward effective conservation in West Africa. The coupling of these results with the human uses of mangroves should be a target in future biomonitoring initiatives in the country and the broader region.