Telomere length (TL) and mitochondrial DNA content (mtDNAc) are biomarkers of biological ageing that respond to multiple stressors, including air pollution. Despite growing research interest, the association between recent and chronic air pollution and these biomarkers in the general population remains unclear. This study investigated the association between air pollution exposure and TL and mtDNAc using data from the 2018 Belgian Health Examination Survey. Multivariable adjusted generalised linear mixed models were applied to assess the exposure to nitrogen dioxide (NO2), fine particulate matter ≤2.5 μm (PM2.5), and black carbon (BC) over 1-week (recent) and 1-year (chronic) periods prior to participation, estimated with a high-resolution spatiotemporal model. Leucocyte TL and mtDNAc were measured using qPCR. A total of 756 participants (mean age 50.6 years, 49.9 % women) were included in the study. Recent exposure to PM2.5 was associated with a 2.40 % (95 % CI: 0.16, 4.69; p = 0.036) longer TL per IQR increment. Trends of lower mtDNAc were observed for chronic exposure to BC (−3.11, 95 % CI: -6.19, 0.07; p = 0.055) and NO2 (−4.02, 95 % CI: -8.22, 0.36; p = 0.072) per IQR increment. No significant associations were observed between chronic air pollution and TL or recent exposure and mtDNAc. These results suggest an inverse association between chronic air pollution and mtDNAc, and a positive association between recent exposure and TL, providing insight into the time-sensitive and air pollutant effects on ageing biomarkers.