A method is presented to evaluate in vitro the efficacy of antibiotics to treat bacteria growing as discrete colonies on surfaces and the contribution of the colony structure to the antibiotic susceptibility of bacteria. Using this method, we explored the relative efficacy of six bactericidal and three bacteriostatic antibiotics to inhibit the growth and kill Staphylococcus aureus colonies of different sizes, densities and ages. As measured by the reduction in viable cell density relative to untreated controls, of the bactericidal drugs tested ciprofloxacin and gentamicin were most effective. By this criteria, ampicillin was more effective than oxacillin. Daptomycin and vancomycin were virtually ineffective for treating S. aureus growing as colonies. The bacteriostatic antibiotic tested, tetracycline, linezolid and erythromycin were all able to prevent the growth of S. aureus colonies and did so even more effectively than daptomycin, which is highly bactericidal in liquid culture. The results of these experiments and other observations suggest that relative inefficacy of oxacillin, vancomycin and daptomycin to kill S. aureus in colonies is due to the density and physiological state of the bacteria rather than the inability of these drugs to penetrate the colonies. The methods developed here are general and can be used to explore the efficacy of antibiotics to treat bacteria growing in biofilms as well as discrete colonies.