Many researchers have advocated in recent times that antiseptic use in healing wounds should be discouraged. Antiseptics have been found to retard healing of wounds. Poloxamer 407 shows thermoreversible properties, which are of the utmost interest in optimizing drug formulation (fluid state at room temperature facilitating administration and gel state above sol-gel transition temperature, at body temperature, promoting prolonged release of pharmacological agents). Chlorhexidine, a commonly used antiseptic, is known to be less toxic on granulation cells. Acting as an antiseptic, it is an effective bactericidal agent against the most categories of microbes, including bacteria, yeast, and viruses. Objective of this study was to evaluate antimicrobial activ- ity of chlorhexidine containing poloxamer gel to Gram-positive and Gram-negative bacteria in vitro. Chlorhexidine gels and chlorhexidine aqueous solutions have different antibacterial activity to S. amis, E.faecalis, E. coli and P. aemginosa strains in vitro. It depends on concentration and dosage form of antiseptic. Study results confirmed that antimicrobial activity of gel depends on active ingredient concentration in antiseptic. The best inhibition effect for both of reference and wild-type bacteria was obtained for 1% chlorhexidine gel. Summarizing the results and assessing the characteristics of the gel ingredients, it can be suggested using chlorhexidine gels in veterinary medicine.