Background: Cutibacterium acnes is part of the anaerobic skin microbiome and resides in deeper skin layers. The organism is an agent of surgical site infections (SSI) in shoulder surgery. We hypothesized that prolonged skin preparation with an agent that penetrates deeply into the skin would be beneficial. Thus, we compared two classes of antiseptics, each combined with alcohol, each applied with two different contact times. Methods: Using a cross-over arrangement, shoulders of 16 healthy volunteers were treated for 2.5 min (standard) or 30 min (prolonged) with alcohol-based chlorhexidine (CHG-ALC) or alcohol-based povidone-iodine (PVP-I-ALC). Skin sites were sampled before, immediately after, and 3 h after treatment, using a standardized cup-scrub technique. Results: Aerobic skin flora was reduced more effectively by PVP-I-ALC than by CHG-ALC after 2.5 min application and immediate sampling (reduction factor [RF] 2.55 ± 0.75 vs. 1.94 ± 0.91, p = 0.04), but not after prolonged contact times and 3-h sampling. Coagulase-negative staphylococci were completely eliminated after PVP-I-ALC application, but still recovered from 4 of 32 samples after CHG-ALC application. Anaerobic flora was reduced more effectively by PVP-I-ALC than CHG-ALC after standard (RF 3.96 ± 1.46 vs. 1.74 ± 1.24, p < 0.01) and prolonged (RF 3.14 ± 1.20 vs. 1.38 ± 1.16, p < 0.01) contact times and immediate sampling, but not after 3-h sampling. No adverse events were reported. Conclusions: PVP-I-ALC showed marginal benefits concerning the aerobic flora, but more substantial benefits over CHG-ALC concerning the anaerobic flora of the shoulder. Standard and prolonged contact times showed superiority for PVP-I-ALC for anaerobic flora at all immediate sampling points, but missed significance at 3-h sampling. The results underscore the need for protection against C. acnes and coagulase-negative staphylococci in orthopaedic surgery. The clinical relevance of these findings, however, should be studied with SSI as an endpoint.