This study presents a new in situ method to explore the impact of macrofauna on seafloor microtopography and corresponding microroughness based on underwater laser line scanning. The local microtopography was determined with mm-level accuracy at three stations colonised by the tubeworm Lanice conchilega offshore of the island of Sylt in the German Bight (south-eastern North Sea), covering approximately 0.5 m2 each. Ground truthing was done using underwater video data. Two stations were populated by tubeworm colonies of different population densities, and one station had 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 independently of the tubeworm density. This effect was not detected by commonly used 1D roughness profiles but required consideration of the complete spectrum. Overall, the results reveal that new indicator variables for benthic organisms may be developed based on microtopographic data. An example demonstrates the use of local slope and skewness to detect tubeworms in the measured digital elevation model.