BACKGROUND: WHO recommends all blood donations be screened for transfusion transmissible infections. However, these data are not incorporated into national surveillance systems in Malawi. We set out to use routinely collected data from blood donors in Blantyre district, Malawi, an area of high HIV and syphilis prevalence, to explore current HIV and syphilis prevalence and identify recent sero-conversions among repeat donors. METHODS: We conducted a retrospective cohort analysis of blood donation data collected by the Malawi Blood Transfusion Service (MBTS) between October 1st 2015 and May 31st 2021. All blood donations were routinely screened for WHO-prioritized transfusion-transmissible infections, including HIV and syphilis. We characterized donor demographics as well as screening outcomes, including identifying sero-conversions among repeat donors who previously tested negative. Logistic regression was used to model the impact of individual level covariates on the probability of sero-conversion. RESULTS: A total of 93,199 donations from 5,054 donors were recorded, with 7 donors (0.1%) donating a maximum of 24 times. The majority of donors were male (4,294; 85%) and students (3264; 64.6%) at the time of their first donation. Of those screened for HIV and syphilis, 126 (2.5%, 126/5,049) and 245 (4.9%, 245/5,043) tested positive respectively.Among repeat donors who previously tested negative, 87 HIV sero-conversions and 195 syphilis sero-conversions were identified over the study period, indicating an HIV incidence rate of 6.86 per 1,000 person-years and a syphilis incidence rate of 15.37 per 1,000 person-years. Donors who were female or aged 16-19 at the time of first donation had a higher risk of HIV or syphilis sero-conversion. CONCLUSIONS: Routinely collected data from national blood donation services may be used to enhance existing population-level disease surveillance systems, particularly in high prevalence areas. While blood donors are generally considered a low-risk population for HIV and syphilis, we were able to identify and characterise blood donor populations at increased risk of sero-conversion over the study period. This information will provide insight into priority prevention areas in Blantyre district and help to inform targeted interventions for improved prevention, testing and treatment.