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The effect of chloroquine phosphate on the pharmacodyanmic activity of ampicillin trihydrate against Staphylococcus aureus NCTC 6571 was investigated using physicochemical and microbiological assay techniques. The physicochemical method was thin layer chromatography and ultraviolet spectrophotometry in a condition simulating normal body temperature and pH. The microbiological assay compared the zones of inhibition of S. aureus NCTC 6571 by ampicillin trihydrate alone and in combination with chloroquine phosphate using the agar diffusion method. The kill kinetics was determined to give a dynamic assessment of the bactericidal activity of ampicilin trihydrate alone and in combination with chloroquine phosphate at various concentrations. The results showed that the zone of inhibition of S. aureus NCTC 6571 by ampicillin trihydrate in the presence of chloroquine was smaller than that produced by ampicillin alone. The kill kinetics revealed a significant increase in the percentage survivor of the organisms in the presence of chloroquine than with ampicillin alone. The results of the study suggest that there is no chemical interaction between the two drugs in vitro however, an antagonistic pharmacodynamic interaction exists between ampicilin trihydrate and chloroquine phosphate in vitro on S. aureus NCTC 6571.   Key words: Ampicillin trihydrate, chloroquine phosphate, drug-drug interaction, antagonism, antimalarial, antibiotic.