The physical properties of biologically impacted seafloor are poorly known, including the effects of biology on local roughness. We present an in-situ laser line scanning method to determine the impact of macrofauna on seafloor microtopography and roughness. The local microtopography was determined with mm-level accuracy for the sand mason Lanice conchilega offshore of the island of Sylt in the German Bight (south-eastern North Sea). Ground truthing was done using underwater video data. Two stations were populated by tubeworm colonies of different population densities, and one station showed a hydrodynamically rippled seafloor. Tubeworms caused an increased skewness of the microtopography height distribution and an increased root mean square roughness at short spatial wavelengths compared with hydrodynamic bedforms. Spectral analysis of the 2D Fourier transformed microtopography showed that the roughness magnitude increased at spatial wavelengths between 0.020 and 0.003 m, eventually independent of the tubeworm density. This effect was not detected by commonly used 1D roughness profiles but required consideration of the complete spectrum. The microroughness of different blue mussel and oyster shells were determined in the laboratory. The potential impact of roughness on high-frequency acoustic scatter – widely used for marine remote sensing and strongly influenced by seafloor roughness – is discussed based on an extensive multi-frequency multibeam echo sounder dataset recorded in shallow water (< 3m) offshore Sylt Island. The results indicate that new indicator variables for benthic organisms may be developed based on the microroughness of benthic organisms.