The management of skin cancer presents challenges in reconstructive surgery to achieve the best patient outcome and rapid wound healing. The most used antiseptic in pre and post operative care is Povidone Iodine solution. However, valid antiseptic alternatives should be considered. The primary endpoint of our study was to achieve the best healing in patients receiving fluorescein solution versus two comparator arms (Eosin and Povidone-Iodine).182 patients were enrolled and divided into three randomized groups according to the type of antiseptic solution received: Fluorescein (n = 63), Eosin (n= 60), and Povidone-Iodine (n = 59). To assess the endpoint of our study the ASEPSIS score was used to objectively evaluate graft healing. Results of the ASEPSIS score for each group were analyzed. The comparison demonstrated that there was no statistically significant difference among the three groups regarding the endpoint of our study, thus between fluorescein and povidone-iodine (p = 0.89), eosin and fluorescein (p = 0.54), and eosin and povidone-iodine (p = 0.81). Patients responded positively to the use of the Fluorescein antiseptic solution, although no more than the others. This study suggests that this type of antiseptic can be successfully used in skin graft management.