Introduction Diphtheria remains a persistent public health threat, despite widespread vaccination and the availability of diphtheria antitoxin (DAT) for treatment. Although mass immunization campaigns have significantly reduced case numbers globally, recent outbreaks, including those in the WHO Region of the Americas, highlight the risks of incomplete immunization. In Brazil, outbreaks in 2010 and 2015 predominantly affected under-immunized individuals. Maintaining high-quality vaccines and DAT is essential to ensuring adequate immunization coverage and effective treatment. This study evaluates the quality control outcomes of diphtheria vaccines and DAT analyzed by the Brazilian National Control Laboratory (NCL) between 2015 and 2024, examining rejection rates and sample distribution across public (National Immunization Program - PNI) and private sectors. Objectives This retrospective observational study aims to analyze the quality control of diphtheria vaccines and DAT in Brazil, verifying the product profile and rejection rates while correlating them with epidemiological trends. Methods Samples submitted to the NCL (2015–2024) were evaluated through document review and, in most cases, analytical assays, according to Brazilian Pharmacopoeia standards. Vaccine samples were categorized by immunization stage to compare them with epidemiological data on coverage, incidence, and product type. Epidemiological data on immunization coverage, applied doses, and diphtheria case reports were analyzed alongside quality control results. Results Between 2015 and 2024, the NCL analyzed 1475 lots of diphtheria vaccines (1459) and DAT (16) to assess compliance with quality standards. A total of 81.9% of vaccine samples originated from the PNI, while 18.1% were from the private sector. The rejection rate remained very low, with 97.56% of all evaluated samples meeting quality standards. Among non-conforming samples, 2.44% of samples (32 pentavalent and 4 DTP booster vaccine) were considered non-conform due to aggregates adhered to the vial surface during visual inspection, and 0.14% (2 pentavalent vaccines) and 0.07% (DAT) were rejected for subpotency of the diphtheria component and diphtheria antitoxin level, while 0.2% (3 pentavalent) reject for subpotency of the pertussis component. These cases represent an extremely small proportion of the total analyzed. Vaccine coverage peaked at >95% in 2015 but declined after 2020 due to vaccine hesitancy and the COVID-19 pandemic, particularly affecting booster doses in adults. This decline coincides with sporadic diphtheria cases, among individuals aged ≥15 years, possibly linked to decreased booster administration. Conclusions Ensuring the quality of diphtheria vaccines and DAT is essential for effective prophylaxis and treatment. Maintaining high immunization coverage across all age groups, adherence to booster doses, and epidemiological surveillance are crucial. The high compliance rate among tested lots underscores the reliability of Brazil’s quality control processes, reducing the likelihood of low-quality immunobiologicals being distributed. However, declining vaccine coverage threatens diphtheria control, particularly in vulnerable populations due to low booster uptake among adults. Given recent outbreaks, the risk of disease reemergence, and the zoonotic potential of the disease, the importance of a One Health approach to its prevention and control is highlighted. Integrating quality control, epidemiological surveillance, sustained vaccine distribution, public awareness, and strategic DAT stockpiling remains vital for preventing and managing future outbreaks.