Fungi are ubiquitous organisms with a wide distribution in almost all ecosystems, including marine environments. Coastal and estuarine ecosystems remain poorly unexplored as fungal habitats, potentially harbouring a hidden diversity with important ecological roles. During an extensive survey of marine fungi in coastal and estuarine Portuguese environments, a collection of 612 isolates was obtained from water, algae, sponges and driftwood. From these, 282 representative isolates were selected through microsatellite-primed PCR (MSP-PCR) fingerprinting analysis, which were identified based on DNA sequence data. The collection yielded 117 taxa from 38 distinct genera, which were identified using DNA sequence analysis. Overall, fungal community composition varied with host/substrate, but the most abundant taxa in the collection were Cladosporium cladosporioides, Penicillium terrigenum, Penicillium brevicompactum and Fusarium equiseti/incarnatum complex. The occurrence of a high fungal diversity harbouring novel species was disclosed. Through a multilocus phylogeny based on ITS, tub2 and tef1-α sequences, in conjunction with morphological and physiological data, we propose Neoascochyta fuci sp. nov. and Paraconiothyrium salinum sp. nov.