To facilitate the conservation of seafloor habitats and planning of offshore activities, there is a growing need for mapping marine benthos in an effective and efficient way. Acoustic data acquired by multi-beam echosounders (MBES) have been extensively used for large-scale and high-resolution seafloor characterization. A deeper understanding of the relationship between backscatter data and sediment compositions can help to identify the benthos occurrence from the MBES data. With two multi-spectral MBES datasets collected near the western Wadden Sea islands in the North Sea, we investigated the potential of mapping marine benthos through backscatter classification. Two unsupervised classification methods, i.e., Bayesian classification, which mainly exploits the backscatter strength from incident angles larger than 20°, and hierarchical clustering of the backscatter strength at different angular ranges, were employed and the results were compared. The classification results from both methods showed a good correspondence with sediment properties such as the median grain size. Moreover, based on a principal component analysis of bottom sample properties, the hierarchical clustering results indicated a better distinction between contributions from the gravel content and benthos occurrence, e.g., sand mason worm density, than Bayesian classification, through involving the backscatter angular variations. Classification for multiple frequencies, on the other hand, showed little difference regarding the relationship with bottom sample properties. Although the backscatter difference between frequencies was also found to positively correlate with certain sample properties, using multi-spectral features for acoustic classification in this study did not reveal additional information compared to single-frequency classification results.