Background: The effects of early-life exposure to house dust mite allergen and endotoxin on the development of asthma are unclear in the literature. We investigated the association of early-life exposure (0-36 months old) to house dust mite allergen and endotoxin with asthma incidence. Methods: In this novel, large-scale, nationwide birth cohort study, 5,017 participants were randomly selected from those who met the eligibility criteria. House dust was vacuum-sampled from the children’s bed within homes and assayed for the presence of dust mite allergen (Der 1) and endotoxin. The participants were classified into four quartiles (Q1-Q4) according to exposure levels. We defined the incidence of asthma and wheezing using a questionnaire at 12, 24, and 36 months old. Odds ratios (ORs) of the incidence of asthma and wheezing by age in Der 1 and endotoxin exposure level were estimated using logistic regression. Results: The cumulative incidence rates of asthma and wheezing during 0-36 months were 10.4% and 38.1%, respectively. Significant ORs were observed in asthma onset during 12-24 months old, asthma onset during 24-36 months old, and wheezing onset during 0-12 months old in the Q4 Der 1 group. In the Q4 endotoxin group, significant positive associations between endotoxin exposure and asthma (OR 2.00, 95% confidence interval [CI]: 1.03-3.85) and wheezing (OR 1.78, 95% CI: 1.01-3.12) onset during 24-36 months old were found. Conclusions: Our results indicated that high levels of early-life exposure to Der 1 and endotoxin in mattresses may be involved in the development of asthma.