Background: Surgical site infection (SSI) is a common yet serious complication of cervical spine surgery. While initially thought to be clinically insignificant, Cutibacterium acnes (C. acnes) is an important cause of infection. The purpose of this study was to investigate the ability of an hydrogen peroxide (H2O2) application during standard pre-surgical skin preparation to reduce the burden of C. acnes in patients undergoing cervical spine surgery. Methods: This was a retrospective review of prospectively collected data. Subjects were randomly assigned to either standard surgical preparation plus H2O2 (experimental) or without H2O2 (control). Pre-scrub, post-scrub, and dermal cultures were obtained to assess the C. acnes burden after cultures on an aerobic and anaerobic growth medium were held for 21 days. Multivariate analysis was conducted to determine factors associated with presence of C. acnes. Outcome measures included the results of intraoperative cultures and the development of a SSI within 90 days post-operatively. Results: Patients (n=86) undergoing elective two- or three-level fusion via anterior approach were included. Prior to application of the antiseptic solution, 65% (28/43) of the experimental cohort and 77% (33/43) of the control cohort had positive C. acnes cultures (p=0.34). Following application of antiseptic solution, there were no differences in positive C. acnes culture rates between the experimental and control cohorts in the epidermal (30% versus 28%, p=1.00) or dermal (40% versus 42%, p=1.00) cultures. No differences in the rates of C. acnes eradication from pre-antiseptic to post-antiseptic application occurred for epidermal (p= 1.00) or dermal (p= 1.00) skin layers. None of the factors were associated with positive C. acnes epidermal cultures on multivariable logistic regression analysis (p> 0.05). Conclusions: While there is potential for H2O2 to reduce the positive culture rate of C. acnes in cervical spine patients, no difference was seen when compared to standard surgical skin preparation.