Floristic surveys are crucial to the conservation of biodiversity, but the vast majority of such surveys are limited to listing species names, and few take into account the evolutionary history of species. Here, we combine classical taxonomic and molecular phylogenetic (DNA barcoding) approaches to catalog the biodiversity of pteridophytes (ferns and lycophytes) of the Nectandra Cloud Forest Reserve, Costa Rica. Surveys were carried out over three field seasons (2008, 2011, and 2013), resulting in 176 species representing 69 genera and 22 families of pteridophytes. Our literature survey of protected areas in Costa Rica shows that Nectandra has an exceptionally diverse pteridophyte flora for its size. Plastid rbcL was selected as a DNA barcode marker and obtained for >95% of pteridophyte taxa at this site. Combined molecular and morphological analyses revealed two previously undescribed taxa that appear to be of hybrid origin. The utility of rbcL for species identification was assessed by calculating minimum interspecific distances and found to have a failure rate of 18%. Finally we compared the distribution of minimum interspecific rbcL distances with two other areas that have been the focus of pteridophyte molecular surveys: Japan and Tahiti. The comparison shows that Nectandra is more similar to Japan than Tahiti, which may reflect the biogeographic history of these floras.