Excessive nitrogen (N) input is one of the most important causative factors of lake eutrophication, which has aroused increasing public attention in past decades. Estrogen contamination is also an increasing environmental problem in aquatic systems around the world. Although both substances usually co-exist in aquatic ecosystems, many researches have only investigated the influences of either N or estrogen individually on sediment bacterial community and nitrous oxide (N2O) emission. Knowledge regarding the combined effects of N and estrogen is still very limited. In this study, a 30-day laboratory incubation experiment was performed to examine the impacts of different N sources (ammonium and nitrate) combined with 17β-estradiol (E2) on sediment bacterial community. High-throughput 16S rRNA gene sequencing technique was used and N2O emission was measured. The results revealed that the relative abundances of Proteobacteria and Bacteroidetes were higher in nitrate treatment than ammonium treatment. Compared to N treatments, N and E2 combined treatments showed higher relative abundances of Proteobacteria, Bacteroidetes, and Firmicutes, but lower relative abundances of Chloroflexi, Acidobacteria, and Actinobacteria over entire incubation period. At the genus level, the relative abundances of genera Flavobacterium, Pseudomonas, Arenimonas, Novosphingobium, Massilia, Aquabacterium, and Bacillus were enhanced by N treatments and especially N and E2 combined treatments, compared to sediment without addition of N and E2. However, the relative abundances of Sporacetigenium, Gaiella, Desulfatiglans, Nitrospira, and Haliangium were decreased in N treatments. Apart from the changes in bacterial community structure, N2O emission was also influenced by different treatments. Nitrate exerted a more significant positive effect on N2O emission than ammonium, and the cumulative emission of N2O was highest in nitrate and E2 combined treatment. Very low abundances of ammonia monooxygenase (amoA) gene and hydroxylamine oxidase (hao) gene were observed in sediments compared to other genes involved in N cycles (such as nitrate reductase (narG and napA) genes, nitrite reductase (nirB, nirK, and nrfA) genes, and nitric oxide reductase (norB) gene), implying that denitrification rather than nitrification played an important role in sediments. The abundances of napA, nirK, and norB were higher in N and E2 combined treatments, indicating that E2 might provide a carbon source for denitrifiers. Moreover, decrease in the abundance of nitrous oxide reductase (nosZ) gene during the denitrifying process in N and E2 combined treatment might be an important reason for increases of N2O emission. These results indicated that alterations of the bacterial community structure due to the co-existence of N and E2 could also change the abundances of genes involved in N cycle.