Skin barrier is often compromised following ablative fractional carbon dioxide laser (AFCO2 ) therapy for acne scarring. The resultant downtime, even of a few days' duration, can be of significant concern to patients. We evaluated the efficacy and safety of topical 0.5% timolol maleate (TM) for its role in short-term restoration of the skin's biophysical properties after laser treatments. This double-blind, placebo-controlled trial included participants aged 18-50 years with atrophic acne scars for at least 3 months. After undergoing laser therapy, they applied 0.5% TM to one cheek and normal saline to the contralateral cheek (control) for 7 days. Corneometry, transepidermal water loss (TEWL) measurement, colorimetry, and clinical outcome parameters (erythema, edema, crusting, pruritus, and tightness scores) were evaluated at baseline and 48, 96, and 168 hours after AFCO2 treatment. Twenty-five healthy participants completed the study. Most participants had Fitzpatrick skin phototype IV. The TM-treated side showed statistically higher corneometry values and lower TEWL than the control side at every follow-up visit (P < 0.001). The crusting score at 96 hours post-AFCO2 treatment was also significantly better on the TM side. No adverse events occurred during the follow-up period. Application of topical 0.5% TM twice daily improves the skin-barrier function and might promote re-epithelialization after laser procedures. Lasers Surg. Med. © 2020 Wiley Periodicals LLC. © 2020 Wiley Periodicals LLC.