After regulation of pesticides, determination of their persistence in the environment is an important indicator of effectiveness of these measures. We quantified concentrations of two types of systemic insecticides: neonicotinoids (imidacloprid, acetamiprid, clothianidin, thiacloprid, and thiamethoxam) and butenolides (flupyradifurone), in off-crop non-target media of hummingbird cloacal fluid, honey bee (Apis mellifera) nectar and honey, and wildflowers before and after regulation of imidacloprid on highbush blueberries in Canada in April 2021. We found that mean total pesticide load increased in hummingbird cloacal fluid, nectar, and flower samples following imidacloprid regulation. On average, we did not find evidence of a decrease in imidacloprid concentrations after regulation. However, there were some decreases, some increases and other cases with no changes in imidacloprid levels depending on the specific media, time point of sampling and site type. At the same time, we found an overall increase in flupyradifurone, acetamiprid, thiamethoxam and thiacloprid, but no change in clothianidin concentrations. In particular, flupyradifurone concentrations observed in biota sampled near to agricultural areas increased by 2-fold in honey bee nectar, 7-fold in hummingbird cloacal fluid, and 8-fold in flowers after the 2021 imidacloprid regulation. The highest residue detected in this study was flupyradifurone at 665 ng/mL (PPB) in honey bee nectar. Mean total pesticide loads were highest in honey samples (84 ± 10 PPB) followed by nectar (56 ± 7 PPB), then hummingbird cloacal fluid (1.8 ± 0.5 PPB), and least, flowers (0.51 ± 0.06 PPB). Our results highlight that limited regulation of imidacloprid does not immediately reduce residue concentrations while other systemic insecticides, possibly replacement compounds, concurrently increase in wildlife.