Paenibacillus larvae and Melissococcus plutonius are the bacterial agents responsible for American and European foulbrood diseases, respectively, and pose significant economic threats to apiculture worldwide. Early detection of these pathogens is critical for effective disease prevention and management in honeybee colonies. P. larvae forms highly persistent spores that can remain infectious on hive equipment for decades, whereas the non-spore-forming M. plutonius can survive for over a year in contaminated materials. Although various decontamination and treatment strategies are available or under development, reliable methods for quantifying these pathogens are essential for accurately assessing their effectiveness. Culture-based techniques and quantitative PCR (qPCR) are commonly used to detect and quantify foulbrood pathogens in hive-associated samples; however, culturing is labor-intensive and sometimes inconsistent, while qPCR does not distinguish between viable and dead cells. Propidium monoazide (PMA) selectively binds DNA from dead cells and prevents its amplification during qPCR, enabling viable-cell quantification. Despite this advantage, the performance of PMA-qPCR for detecting P. larvae and M. plutonius in hive samples has not previously been assessed. In this study, we evaluated PMA-qPCR using both pure cultures and environmentally relevant honeycomb samples before and after ozone treatment. PMA treatment greatly reduced the erroneous detection of dead cells by several orders of magnitude in both culture and hive samples, supporting its utility for AFB and EFB risk assessment and treatment evaluation. However, incomplete suppression of dead-cell DNA at high cell densities indicates that further optimization of PMA treatment conditions may be necessary.