Bacterial infections cause major complications in wound healing. Local antiseptics are used for daily wound care; however, their potential toxic effects on the vasculature have not yet been thoroughly investigated. The aim of this study was to assess the effects of antiseptics on microcirculation. Investigations were performed on a standardized cremaster muscle model on rats (n = 60). The arteriolar diameter and functional capillary density (FCD) were investigated using transillumination microscopy before and 60 and 120 minutes after application of each of the following antimicrobial agents: alcohol, hydrogen peroxide, imipenem, octenidine dihydrochloride, polyhexanide, and ethacridine lactate. Although polyhexanide caused a significant arteriolar dilatation (106.25 ± 3.23 vs. 88.54 ± 6.74 μm [baseline value]) and increase of FCD compared with baseline value (12.65 ± 0.82 vs. 9.10 ± 0.50 n/0.22 mm), alcohol led to a significant decrease of both parameters (90.63 ± 10.80 vs. 52.09 ± 7.69 and 5.35 ± 0.54 vs. 1.68 ± 0.48) and was the only agent that caused arteriolar thrombosis. The FCD also increased significantly after treatment with hydrogen peroxide (10.55 ± 0.33 vs. 12.30 ± 0.48) and octenidine (6.82 ± 0.63 vs. 12.32 ± 0.63). However, no positive effect on arteriolar diameter could be found. Ethacridine lactate and imipenem did not impact either parameter. In addition to reducing bacteria, an antiseptic should be nontoxic, especially to the microcirculation. Polyhexanide seems to have a positive influence on vessel diameter and capillary density, whereas alcohol reduces both parameters. If the antimicrobial efficacy is comparable, the antiseptic with less toxic effects should be chosen, especially in critically perfused wounds.