DNA replication requires the activity of a class of enzymes called the topoisomerases. Topoisomerase II (DNA gyrase) relaxes supercoiled DNA molecules and initiates transient breakages and rejoins phosphodiester bonds in superhelical turns of closed-circular DNA of bacteria. This allows the DNA strand to be replicated by DNA or RNA polymerases. Quinolones are a key group of antibiotics that specifically interfere with bacterial topoisomerase II and not mammalian topoisomerases. Fluoroquinolones, second-generation quinolones that include levofloxacin, norfloxacin, and ciprofloxacin, are active against both Gram-negative and Gram-positive bacteria. Inhibitors that are effective against mammalian topoisomerases, such as irinotecan and etoposide, are used as antineoplastic drugs to kill cancer cells.

Some antibiotics specifically interfere with RNA synthesis by inhibiting RNA polymerases. Rifampicin blocks initiation of RNA synthesis by specifically inhibiting bacterial RNA polymerase. It does not interact with mammalian RNA polymerases, making it specific for Gram-positive bacteria and some Gram-negative bacteria. Antibiotics such as doxorubicin and actinomycin D (dactinomycin), are not specific for bacteria and interfere with both bacterial and mammalian systems. These are most often used as antineoplastic and antitumor drugs, attacking rapidly growing malignant cells as well as normal cells. Because cancerous cells are growing at a faster rate than surrounding normal tissue, a higher percentage of malignant cells are attacked by cytotoxic drugs.