Aim: In this study, we assessed the importance of local- to landscape-scale effects of land cover and land use on flying insect biomass. Location: Denmark and parts of Germany. Methods: We used rooftop-mounted car nets in a citizen science project (“InsectMobile”) to allow for large-scale geographic sampling of flying insects. Volunteers sampled insects along 278 five-km routes in urban, farmland, grassland, wetland and forest landscapes in the summer of 2018. The bulk insect samples were dried overnight to obtain the sample biomass. We extracted proportional land use variables in buffers between 50 and 1,000 m along the routes and compiled them into land cover categories to examine the effect of each land cover, and specific land use types, on insect biomass. Results: We found a negative association between urban cover and flying insect biomass (1% increase in urban cover = 1% [95% CI: −3.0 to 0.0] decrease in biomass in Denmark, and a 3% [95% CI: −3.0 to 0.0] decrease in Germany) at a landscape scale (1,000-m buffer). In Denmark, we also found positive effects of semi-natural land cover types, that is protected grassland (largest at the landscape scale, 1000 m) and forests (largest at intermediate scales, 250 m). Protected grassland cover had a stronger positive effect on insect biomass than forest cover did. For farmland cover, the positive association with insect biomass was not clearly modified by any variable associated with farmland use intensity. The negative association between insect biomass and urban land cover appeared to be reduced by increased urban green space. Main conclusions: Our results show that land cover has an impact on flying insect biomass with the magnitude of this effect varying across spatial scales. However, the vast expanse of grey space in urbanized areas has a direct negative impact on flying insect biomass across all spatial scales examined.