Acoustic backscatter has been used as a tool to map the seafloor in greater detail and plays an increasingly important role in seafloor mapping to meet multiple ocean management needs. An outstanding challenge to the use of backscatter for seafloor mapping is the distinction between acoustically similar substrates, such as mixed sediments from rhodoliths. Rhodolith beds are a biogenic substrate that provides important ecological services, and are typically classified as a single categorical substrate type—though nodules coverage may be spatially variable. Recently, multispectral acoustic backscatter has demonstrated great potential to improve thematic seafloor mapping compared to single-frequency systems. This work employs multispectral multibeam backscatter and underwater imagery to characterize and map rhodolith beds in the Costa das Algas Marine Protected Area (Brazil). A support vector machine classifier was used to classify multifrequency backscatter mosaics according to rhodolith classes identified from underwater imagery. Results suggest that multispectral backscatter is effective both in providing information for mapping different proportions of rhodolith coverage and in predicting the presence or absence of these nodules. The backscatter of the lowest frequency was the most useful for distinguishing variable proportions of rhodolith coverage, and the two higher frequencies were better predictors of presence and absence.