Abstract Background Prior studies in humans have suggested that telomere shortening may be accelerated by infection, but research on multiple pathogens and use of large population-based study samples has been limited. We estimated cross-sectional associations between seropositivity to five persistent pathogens (Herpes Simplex Virus Type-1 (HSV-1), Herpes Simplex Virus Type-2 (HSV-2), cytomegalovirus (CMV), Helicobacter pylori (H.pylori), and Hepatitis B) as well as total pathogen burden and leukocyte telomere length. Data were derived from the National Health and Nutrition Examination Survey (1999–2000) for individuals 20–49 years of age, N = 1708. We analyzed the influence of each pathogen separately, a pathogen count score and a latent class model of pathogen burden on log telomere length using linear regression models, adjusted for covariates. Results Individuals in a latent pathogen burden class characterized by high probabilities of infection with HSV-1, CMV, and H. pylori, had significantly decreased log telomere length (− 0.30 [95% CI: − 0.36, − 0.24]) compared to those in a latent class characterized by low probabilities of all five infections. There were limited significant associations using other pathogen measures. Conclusions These results suggest that infection with specific combinations of pathogens may be one mechanism contributing to accelerated cellular senescence with possible origins early in the life course.