Cefotaxime is used for infections of the respiratory tract, skin, bones, joints, urogenital system, meninges, and bloodstream. It generally has good coverage against most Gram-negative bacteria, with the notable exception of Pseudomonas. It is also effective against most Gram-positive cocci except for Enterococcus.^[1] It is active against penicillin-resistant strains of Streptococcus pneumoniae. It has modest activity against the anaerobic Bacteroides fragilis. In meningitis, cefotaxime crosses the blood–brain barrier better than cefuroxime.

It inhibits bacterial cell wall synthesis by binding to one or more of the penicillin-binding proteins (PBPs). This inhibits the final transpeptidation step of peptidoglycan synthesis in bacterial cell walls, thus inhibiting cell wall biosynthesis. Bacteria eventually lyse due to ongoing activity of cell wall autolytic enzymes (autolysins and murein hydrolases) while cell wall assembly is arrested.^[1]

Cefotaxime, like other β-lactam antibiotics does not only block the division of bacteria, including cyanobacteria, but also the division of cyanelles, the photosynthetic organelles of the Glaucophytes, and the division of chloroplasts of bryophytes. In contrast, it has no effect on the plastids of the highly developed vascular plants. This supports the endosymbiotic theory and indicates an evolution of plastid division in land plants. ^[2]