Piperacillin is a broad-spectrum β-lactam antibiotic of the ureidopenicillin class. The chemical structure of piperacillin and other ureidopenicillins incorporates a polar side chain that enhances penetration into Gram negative bacteria and reduces susceptibility to cleavage by Gram negative beta lactamase enzymes. These properties confer activity against the important hospital pathogen Pseudomonas aeruginosa. Thus piperacillin is sometimes referred to as an "anti-pseudomonal penicillin".

When used alone, piperacillin lacks strong activity against the Gram positive pathogens such as Staphylococcus aureus, as the beta-lactam ring is hydrolyzed by the bacteria's beta-lactamase

Piperacillin is most commonly used in combination with the beta-lactamase inhibitor tazobactam (Piperacillin/Tazobactam) (tradename "Zosyn" or "Tazocin"), which enhances piperacillin's effectiveness by inhibiting many beta lactamases to which it is susceptible. The co-administration of tazobactam does not confer activity against MRSA, however, as penicillins (and most other beta lactams) do not avidly bind to the penicillin-binding proteins of this pathogen.