Improvements to acoustic seafloor mapping systems have motivated novel marine geological and benthic biological research. Multibeam echosounders (MBES) have become a mainstream tool for acoustic remote sensing of the seabed. Recently, “multispectral” MBES backscatter, which is acquired at multiple operating frequencies, has been developed to characterize the seabed in greater detail, yet methods for the use of these data are still being explored. Here, we evaluate the potential for seabed discrimination using multispectral backscatter data within a multi-method framework. We present a novel MBES dataset acquired using four operating frequencies (170, 280, 400, and 700 kHz) near the Doce River mouth, situated on the eastern Brazilian continental shelf. Image-based and angular range analysis methods were applied to characterize the multifrequency response of the seabed. The large amount of information resulting from these methods complicates a manual seabed segmentation solution. The data were therefore summarized using a combination of dimensionality reduction and density-based clustering, enabling hierarchical spatial classification of the seabed with sparse ground-truth. This approach provided an effective solution to synthesizing these data spatially to identify two distinct acoustic seabed classes, with four subclasses within one of the broader classes, which corresponded closely with seafloor sediment samples collected at the site. The multispectral backscatter data also provided information in likely, unknown, sub-surface substrate differences at this site. The study demonstrates that the adoption of a multi-method framework combining image-based and angular range analysis methods with multispectral MBES data can offer significant advantages for seafloor characterization and mapping.