Background Acute bouts of exercise have been shown to improve motor learning. However, whether these benefits can be observed from habitual physical activity (PA) levels remains unclear and has important implications around PA guidelines to promote motor learning across the lifespan. This study investigated the effect of habitual PA levels on brain activity within the dorsolateral prefrontal cortex (DLPFC) during procedural motor skill acquisition. Methods Twenty-six right-handed healthy young adults had physical activity levels quantified by calculating the metabolic equivalent of task (METs) in minutes per week, derived from the International Physical Activity Questionnaire (IPAQ). Functional near-infrared spectroscopy (fNIRS) over the DLPFC was recorded to measure neural activation during a serial reaction time task (SRTT). Behavioural indicators of procedural motor skill acquisition were quantified as reaction time and accuracy of correct trials during the SRTT. DLPFC activation was characterised as task-related changes in oxyhaemoglobin (∆[HbO2]). Results Findings showed that higher PA levels were associated with improvements in reaction time during procedural motor skill acquisition (p = 0.03). However, no significant effects of PA levels on accuracy or ∆[HbO2] during procedural motor skill acquisition were observed. These findings show that while habitual PA may promote motor performance in young adults, this is not reflected by changes in the DLPFC area of the brain.