Background <p>Polypharmacy is associated with reduced health-related quality of life (HRQoL). This study explores the association between prescription and non-prescription medication pill burdens, independent of underlying morbidity, on HRQoL in an older adult population.</p> Methods <p>Data from the final intervention year of the ASPirin in Reducing Events in the Elderly (ASPREE) randomized trial in older adults from Australia and the USA, were analyzed cross-sectionally.&#xa0;Participants reported daily prescription and non-prescription pill counts at the final trial visit. HRQoL was assessed using the 12-Item Short-Form instrument (SF-12) and summarized into the physical component summary (PCS) score and mental component summary (MCS) score, where lower scores reflect poorer HRQoL. Multivariable regression, adjusted for covariates, was used to examine the relationships of categorized prescription and non-prescription pill counts with PCS and MCS separately.</p> Results <p>15,165 participants responded to the question about prescription use and 15,727 for non-prescriptions (mean age = 80 years). Compared with non-users of prescription medications, lower mean PCS scores and larger reductions in scores were seen as prescription medication pill burden increased from 1–3, 4–6, 7–9, to ≥ 10 pills (−&#xa0;1.7, −&#xa0;4.5, −&#xa0;7.6, and −&#xa0;10.9, respectively, <i>p</i> &lt; 0.001). A similar relationship, but of lesser magnitude, was observed with non-prescription medication pill burden, where the mean PCS was lower by −&#xa0;0.2 for 1–3 pills (<i>p</i> = 0.494), −&#xa0;1.8 for 4–6 (<i>p</i> &lt; 0.001), and −&#xa0;1.9 for ≥ 7 pills (<i>p</i> &lt; 0.001), compared with non-users. No significant association was observed between prescription or non-prescription medication pill burdens and MCS.</p> Conclusions <p>Prescription and non-prescription medication pill burdens are independently associated with reduced physical, but not mental, HRQoL in older adults.</p>