The aim of the present study was to analyze if small-scale spatial variation of benthic diatom assemblages has consequences for biomonitoring. Benthic diatom samples were collected at one sampling site in a mesotrophic stream in Middle-Sweden from stone and plant (macrophytes and mosses) substrate. Our results showed that spatial variation of both the diatom species composition and the calculated bioindices were similar on both small (distance of centimeter) and medium (distance of decimeters) scales. Spatial variation was also similar on both studied substrates. This implies that it does not matter if a small or a larger area is sampled for biomonitoring as long as no major environmental factors impact certain sites systematically. Diatom assemblages and indices were significantly different between substrates. Spatial variation did not contribute much to this variation, and variation on a slide was unimportant. These results confirm earlier findings that smallscale spatial variation is not a problem when using diatoms to detect anthropogenic impacts to a stream or lake.