In vitro, thienamycin employs a similar mode of action as penicillins through disrupting the cell wall synthesis (peptidoglycan biosynthesis) of various Gram-positive and Gram-negative bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa to name a few).    Although thienamycin binds to all of the penicillin-binding proteins (PBPs) in Escherichia coli, it preferentially binds to PBP-1 and PBP-2, which are both associated with the elongation of the cell wall.
Unlike penicillins, which are rendered ineffective through rapid hydrolysis by the β-lactamase enzyme present in some strains of bacteria, thienamycin remains antimicrobially active.  Thienamycin displayed high activity against bacteria that were resistant to other β-lactamase-stable compounds (cephalosporins), highlighting the superiority of thienamycin as an antibiotic among β-lactams.