Chlorhexidine is a bactericidal antiseptic effective against a large range of vegetative gram positive and gram negative bacteria, although more effective against gram positive bacteria. Some species of Pseudomonas and Proteus are relatively less susceptible.

Chlorhexidine is ineffective against acid-fast bacteria, bacterial spores, fungi and viruses. As a cationic material, chlorhexidine is incompatible with soaps and other anionic materials. At a concentration of 4% chlorhexidine is incompatible with borates, bicarbonates, carbonates, chlorides, citrates, phosphates and sulfates, forming salts of low solubility which may precipitate from solutions after standing.

Chlorhexidine is most active at a neutral pH; its activity is reduced by organic matter (blood, serum, etc.) and by phospholipids.