<p>Previous studies indicate that route familiarity can adversely affect drivers’ attentional states. However, few studies have examined how route familiarity affects attentional resource allocation using physiological measures. We investigated the effect of route familiarity on attentional resource allocation by measuring physiological indices during real-world road driving. Participants (<i>n</i> = 10) drove a vehicle on public roads on the same route over six days. We analyzed the eye-fixation-related brain potentials (EFRPs), auditory-evoked potentials (AEPs), and eye-blink duration from the electroencephalographic (EEG) and electrooculographic (EOG) signals as indices of attentional resource allocation. The results indicated that the P2 amplitude of the AEPs elicited by task-irrelevant auditory probes significantly increased on Days 3–4 and Days 5–6 compared to Days 1–2, suggesting that attentional resources allocated to cognitive processing decreased as route familiarity increased. The P1 amplitude of the EFRPs decreased across days, with a significant reduction from Days 1–2 to Days 3–4, suggesting that attentional resources allocated to visual processing also decreased as route familiarity increased. This study supports previous findings that route familiarity affects drivers’ attentional states, providing new objective physiological evidence. We also discussed the differences in drivers’ inattention caused by route familiarity compared to the driving duration.</p>