Mycoplasmas are the smallest free-living microorganisms, being about 300 nm in diameter. They are bounded by a triple-layered membrane and, unlike conventional bacteria, do not have a rigid cell wall. Hence, they are not susceptible to penicillins and other antibiotics that act on this structure. They are, however, susceptible to a variety of other broad-spectrum antibiotics, most of which only inhibit their multiplication and do not kill them. The tetracyclines have always been in the forefront of antibiotic usage, particularly for genital tract infections, but macrolides are also widely used for respiratory tract infections. Indeed, in comparison with the tetracyclines, erythromycin, the newer macrolides, the ketolides and the newer quinolones have equal or sometimes greater activity. The two latter antibiotic groups also have some cidal activity. The antibiotic susceptibility profiles of several mycoplasmas of human origin are presented, those of Mycoplasma pneumoniae and Mycoplasma genitalium being similar. Apart from the penicillins, mycoplasmas are innately resistant to some other antibiotics, for example the rifampicins. In addition, some may develop resistance, either by gene mutation or by acquisition of a resistance gene, to antibiotics to which they are usually sensitive. Resistance of mycoplasmas to tetracyclines is common and due to acquisition of the tetM gene. The antibiotic susceptibility pattern may be influenced greatly by the source of the mycoplasma; for example, one recovered from a contaminated eukaryotic cell culture that has been subjected to extensive antibiotic treatment may have an antibiotic profile quite different from the same mycoplasmal species that has been recovered directly from a human or animal source. Mycoplasmas may be difficult to eradicate from human or animal hosts or from cell cultures by antibiotic treatment because of resistance to the antibiotic, or because it lacks cidal activity, or because there is invasion of eukaryotic cells by some mycoplasmas. Eradication may be particularly difficult in immunosuppressed or immunodeficient individuals, particularly those who are hypogammaglobulinaemic. The regimes that are most likely to be effective in the treatment of respiratory or genitourinary mycoplasmal infections are presented.