Objective To analyze the impact of different infection sources on the mortality risk of patients with secondary bloodstream infection (BSI), and provide reference for precise prevention and treatment for secondary BSI. Methods Clinical data of 340 patients with secondary BSI at the First Affiliated Hospital with Nanjing Medical University from January 2020 to December 2024 were collected for a retrospective cohort study. According to the sources of infection, patients were divided into abdominal infection group, lower respiratory tract infection group, urinary tract infection group, and other infection group. The impact of different infection sources on the mortality risk of patients with secondary BSI was analyzed using COX regression model. Results The sources of secondary BSI from high to low were abdominal infection (n=135, 39.71%), lower respiratory tract infection (n=105, 30.88%), urinary tract infection (n=68, 20.00%), and other infection (n=32, 9.41%). The mortality of secondary BSI from different infection sources from high to low were as follows: lower respiratory tract infection (59.05%), other infection (34.38%), abdominal infection (27.41%), and urinary tract infection (10.29%). Multivariate COX regression analysis result showed that patients with secondary BSI from lower respiratory tract infection had a higher risk of mortality than those from abdominal infection (HR=2.22, 95% CI [1.34-3.67], P=0.002), urinary tract infection (HR=4.10, 95% CI [1.79-9.40], P<0.001), and other infection sources (HR=2.26, 95% CI [1.01-5.04], P=0.046). Conclusion There are differences in the mortality risk of patients with secondary BSI from different infection sources. Lower respiratory tract infection is higher than abdominal infection, urinary tract infection, and other infection sources. Strengthening the prevention and control of lower respiratory tract infection and multidrug-resistant organism (MDRO) infection, and paying attention to the diagnosis and treatment of secondary BSI from lower respiratory tract infection source can help to reduce the mortality risk of patients.