Polymyxin B is used for infections with gram-negative organisms, but may be neurotoxic and nephrotoxic. All gram-positive bacteria, fungi, and the gram-negative cocci, are resistant.

Polymixin B acts against microorganisms by binding to and interfering with the permeability of the cytoplasmic membrane. After binding to lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria, polymyxins disrupt both the outer and inner membranes. The hydrophobic tail is important in causing membrane damage, suggesting a detergent-like mode of action.

Polymixin B can be used with a basal medium and additional antibiotics to select specific microorganisms like Acidovorax avenae and Ralstonia solanacearum for phytopathology.