Précis: Femtosecond laser-assisted cataract surgery (FLACS) may cause thinning of the peripapillary retinal nerve fiber layer (pRNFL) in healthy eyes. Purpose: This prospective cohort study aimed to compare changes of pRNFL after FLACS using a liquid patient interface and conventional phacoemulsification cataract surgery (CPCS). Patients and Methods: Included were 261 eyes (261 patients) with age-related cataracts and no ocular diseases scheduled either for FLACS (222 eyes) or CPCS (39 eyes). FLACS was performed using a Ziemer LDV Z8 laser. Average and quadrant pRNFL thickness was measured using optical coherence tomography before surgery and at 1, 3, and 6 months postoperatively. Postoperative changes in pRNFL thickness were compared within and between groups. Results: Mean quadrant and average pRNFL thicknesses significantly increased after both surgeries (P<0.001). However, pRNFL thinning occurred after FLACS and CPCS (17% vs. 5.1%, respectively, P>0.05). FLACS eyes showed a significant and stable decrease of average pRNFL thickness (P=0.057) and a gradual decrease in pRNFL thickness of all quadrants (P≤0.018). CPCS eyes showed an initial increase of pRNFL thickness, followed by a decrease only in the nasal quadrant and average pRNFL. Preoperative pRNFL thickness was associated with thinning of the temporal quadrant (P=0.04). Conclusions: Both FLACS and CPCS demonstrated pRNFL thinning in some healthy eyes. Although the higher rate of pRNFL thinning after FLACS compared with CPCS lacked statistical significance, a consistent decrease in pRNFL thickness occurred in all quadrants and average pRNFL of FLACS eyes, suggesting that FLACS may lead to pRNFL thinning. Eyes with thinner preoperative pRNFL may be prone to temporal quadrant thinning after FLACS.