Previously, we described methods for measuring in vivo antimicrobial activity in which the resident bacterial flora of the forearm is expanded by occlusion with an impermeable plastic film, test agents are applied and quantitative cultures are obtained at varying time points. This methodology allows for an in vivo quantitative assessment of antimicrobial effects directed against a dense flora comprised primarily of staphylococci. This method may not be applicable to situations in which there is a high density of multiple species of bacteria. We describe herein new methods which permit in vivo determination of antimicrobial activity against a dense, mixed flora. Swabs moistened with a dilute nonionic detergent are used to remove bacteria from the subject’s axilla or groin which are then translocated to the subject’s forearm. Occlusion of the forearm with a large, sterile plastic chamber provides the necessary humid environment to yield a dense flora (105–106 CFU) consisting of gram-positive cocci, gram-positive coryneforms and gram-negative rods. In this manner, multiple test sites are created on each forearm allowing for the simultaneous evaluation of multiple antimicrobial agents in a single subject. This method allows for the evaluation of the immediate, as well as sustained, in vivo bactericidal effect of an antimicrobial agent against a dense mixed flora with quantitative cultures obtained at varying time points after application of the test agent. Furthermore, ecological pressures which select for resistant organisms or allow for an overgrowth of nonsensitive bacteria can be evaluated by determining the composition of the flora after single or repeated applications of a test agent. The testing methodologies described herein can provide relevant information regarding the antimicrobial effectiveness of an agent in a variety of situations such as use against the axillary flora (including its utility as a deodorant), use as a perineal cleanser for critically ill, hospitalized patients and use in situations where a dense mixed flora exists, e.g. stasis ulcers and infected intertriginous dermatoses.