The Cabo Rojo Solar Salterns located on the southwest coast of Puerto Rico are composed of two main ecosystems (i.e., salt ponds and microbial mats). Even though these locations are characterized by high solar radiation (mean light intensity of 39 mol photons m-2d-1) they harbour a diverse microscopic life. We used morphological and molecular techniques to identify a series of halotolerant fungi. A total of 183 isolates and 36 species were cultured in this study. From the water from the salt ponds, 86 isolates of 26 species were cultured. The halotolerant fungi isolated from water were: Cladosporium cladosporioides, nine Aspergillus sp., five Penicillium sp. and the black yeast Hortaea werneckii. A distinctive isolate with a blue mycelium was cultured from the salt ponds, representing a new species of Periconia based on morphology and rDNA analysis. Forty-four isolates from eight species were cultured from the sediments around the salt ponds. Most of the sediment isolates formed only sterile mycelium, while several were Chaetomium globosum. A total of 53 isolates from 16 species were isolated from the three layers of the microbial mats, of which Aspergillus niger was the most frequent isolate. Phospholipid fatty acid profiles generated from the different layers of the microbial mats indicated that the uppermost layers of the mats contained fungal biomarker, 18:2w6. This fatty acid decreased with depth, the highest concentration was observed in the green upper layer and it disappeared in the black bottom anoxic layer. This correlates with the isolation of fungi using the serial dilution technique. This is the first study that documents the presence of fungi in microbial mats.