Biosolids are applied to agricultural land as a soil conditioner and source of crop nutrients. However, there is concern that bacteria from biosolids may become established in soils, particularly if that soil becomes water-logged. This study examined the microbial community of arable soils cultivated with barley under different applications of biosolids (0, 24t/ha, 48t/ha) in laboratory mesocosms which simulated a 10-day flood. Nutrients (P and N) and organic matter in the soil increased with application rate, but plant growth was not affected by biosolid application. The biosolids contained 10× more genetic material than the soil, with much lower bacterial diversity, yet application did not significantly change the taxonomy of the soil microbiome, with minor changes related to increased nutrients and SOM. Anaerobic conditions developed rapidly during flooding, causing shifts in the native soil microbiome. Some bacterial taxa that were highly abundant in biosolids had slightly increased relative abundance in amended soils during the flood. After flooding, soil bacterial populations returned to their pre-flood profiles, implying that the native microbial community is resilient to transient changes. The short-term changes in the microbiome of biosolid-amended soils during flooding do not appear to increase the environmental risk posed by biosolid application.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11356-023-27424-0.