Overwintering benthic algae not only directly impact drinking water safety, but also affect the algae recruitment in warm spring seasons. Thus, understanding the characteristics of overwintering benthic algae can provide scientific references for formulating preventative strategies of reasonable water resource. However, they have received less attention. In this study, the spatiotemporal variation of benthic algae and their harmful secondary metabolites were studied from autumn to summer in Qingcaosha Reservoir. Benthic algae (picophytobenthos accounting for 55.42%) had a high biomass during overwintering, and the groups of overwintering benthic algae included pico-Cyanobacteria, pico-Cryptomonas, pico-Chlorophyta, pico-Diatoms, Cyanobacteria, Chlorophyta, Cryptomonas and Diatoms, which were consistent with the planktonic algae species in warm seasons. In oligotrophic or mesotrophic water bodies, micronutrients of iron and manganese were key nutrient factors influencing the biomass of benthic algae. Furthermore, picophytobenthos were important potential contributors of harmful secondary metabolites. The content of microcystins, anatoxin-a, geosmin and 2-methylisoborneol in sediment were 15.75 µg/kg·FW, 48.16 µg/kg·FW, 3.91 ng/kg·FW, and 11.76 ng/kg·FW during winter, which had potential to be released into water bodies to impact water quality. These findings indicate that water quality monitoring programs need to consider sediment in winter as a potential source of toxins and preventative measures to prevent excessive proliferation of algae should be implemented in winter.