Wound contaminants are the main cause of healing delay and infection in both chronic and acute wounds; for this reason, the microbial infection management in wound healing is one of the most important components for an effective standard of care. The wound contaminants are most likely to originate from the environment and from the surrounding skin lesion, and to date, the most frequent bacteria isolated are Staphylococcus aureus, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In order to counteract and control these contaminants, the standard care includes topical antiseptic agents. The most commonly used include iodine-releasing agents (e.g., povidone-iodine), hydrogen peroxide, and polyhexanide. This study aims to investigate the in vitro antibacterial activity of a novel topical spray (Fitostimoline® Plus spray) based on 0.1% polyhexanide and Rigenase® against S. aureus, P. aeruginosa, K. pneumoniae, and the combination of S. aureus and K. pneumoniae. The in vitro antimicrobial activity of Fitostimoline® Plus spray was evaluated by the agar disk diffusion assay, quantitative suspension test, and quantitative carrier test, demonstrating that Fitostimoline® Plus spray is able to kill 99.9% bacteria. These results support the microbiological characterization of Fitostimoline® Plus spray confirming the antibacterial activity of polyhexanide (PHMB).