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Background Although WHO recommends that all blood donations be screened for transfusion transmissible infections, these data are currently not incorporated into national surveillance. We set out to use routinely collected data from blood donors in Blantyre district, Malawi to explore HIV and syphilis prevalence and identify seroconversions among repeat donors. Methods We conducted a retrospective cohort analysis of blood donation data collected by the Malawi Blood Transfusion Service (MBTS) from October, 2015 to May, 2021. All blood donations were routinely screened for HIV and syphilis. We characterized donor demographics as well as screening outcomes, including identifying seroconversions among repeat donors who previously tested negative on their last donation. Log binomial regression was used to model the impact of individual level covariates on HIV and syphilis prevalence and risk of seroconversion. Results A total of 23,280 donations from 5,051 donors were recorded, with a median frequency of donations of 3 (IQR:2–6). At the time of their first donation, most donors were male (4,294; 85%) and students (3262; 64.6%). Of those screened for HIV and syphilis at first donation, 1.0%, (52/5,051) and 1.6% (80/5,051) tested positive, respectively. Among repeat donors who previously tested negative, 52 HIV seroconversions and 126 syphilis seroconversions were identified, indicating an incidence rate per 1,000 person-years of 5.9 (95% CI: 4.7, 7.4) and 13.3 (95% CI:11.4, 15.4) respectively. Prevalence of HIV (Prevalence ratio (PR) = 0.31: 95% CI: 0.15, 0.65) and syphilis (PR = 0.54: 95% CI:0.30, 0.94) were lower and risk of syphilis seroconversion (Risk ratio = 0.47: 95% CI:0.31, 0.70) was higher among students compared to other donors. Conclusions While blood donors are generally considered a low-risk population for HIV and syphilis, we were able to identify that there remain relatively high rates of undiagnosed HIV and syphilis infections among donors. The routinely collected data from national blood donation services may be used to better understand the epidemiology of HIV and syphilis in specific locations, with the potential to enhance existing population-level disease surveillance systems. In addition, these findings may be used to identify priority prevention areas and populations in Blantyre district that can inform targeted interventions for improved disease prevention, testing and treatment.