Skin and soft tissue infections (SSTIs) are very common in dermatology and the use of antimicrobial formulations are important in treating these diseases. With the increasing of drug-resistant strains, researchers need to find ways to enhance the effectiveness and/or reduce the drug resistance. Clioquinol was one of antiseptics that can inactivate microbes. It was lack of data of antimicrobial activity; meanwhile it was infrequently used in infection. In order to research the antimicrobial spectrum and activity of topical 3% clioquinol cream among common pathogenic microorganisms compared with other common topical pharmaceuticals, we used modified agar diffusion assay to judge drug susceptibility and compared with broth microdilution assay. Thirty strains of pathogenic fungi belonging to 14 species and 5 strains of pathogenic bacterium belonging to 4 species from clinic or standard strains were enrolled into the experiment. The inhibition zone around 3% clioquinol cream for all experiment isolates was observed. It could inhibit the growth of most fungal species with different strength, but the antibacterial activity was weak. For Candida tropicalis, Candida guilliermondii, Aspergillus terreus, Fusarium solani and Trichoderma harzianum, the inhibition zone was biggest among all the tested drugs. The antifungal activity for Dermatophytes and Candida albicans was moderate. Two assays had a degree of consistency. Based on results above, we identified the antifungal spectrum of 3% clioquinol cream was broad. The antimicrobial strength of 3% clioquinol cream depended on the species but it can act on most of the species.