Algae are a group of highly diverse photosynthetic organisms found in variety of habitats. As the primary energy base in ecosystems, knowledge of the diversity and presence of certain algal lineages is paramount to our understanding of the trophic state of aquatic habitats. In recent years, the state of Florida has seen an increase of both marine and freshwater algal blooms. Similarly, filamentous algae have begun outcompeting vascular macrophytes throughout many of Florida’s springs as nutrient enrichment from anthropogenic sources increases. Traditionally, the Florida algal spring communities have been assessed using classic morphological methods, which may underrepresent the true biodiversity present. Therefore, the goal of this study was to conduct a more complete diversity assessment implementing next-generation sequencing techniques (NGS) with morphological analyses and culturing methods. While morphological methods identified a wide variety of algal taxa, belonging to 4 phyla (Bacillariophyta, Charophyta, Chlorophyta, and Cyanobacteria), next-generation sequencing techniques provided greater detail of the diatom community. This is particularly important as many diatom taxa are used as indicators of water quality. We noted discrepancies between these two methods, highlighting how NGS techniques may complement the use of morphological analyses when analyzing algal diversity in this system. Culturing methods also revealed the presence of two taxa new to science (Nodosilinea fontisand Brasilonema variegatus), indicating these springs may represent a potential source of novel cyanobacteria. Taken together, this study showcases Florida springs are rich in algal diversity and a combination of methods is required for more complete biodiversity assessments. Future studies implementing such methods will aid in the preservation and conservation of these ecosystems.