The antibacterial activity of anodic porous alumina (APA) prepared using phosphoric, oxalic, and sulfuric acids was tested, and it was found that APA prepared using sulfuric acid had the highest antibacterial activity against Staphylococcus aureus and Escherichia coli. In addition, the results of the antibacterial test using APA prepared with different concentrations of sulfuric acid showed that APA prepared in concentrated sulfuric acid exhibited significantly higher antibacterial activity. APA formed in concentrated sulfuric acid also showed excellent antibacterial activity even at a high culture medium concentration, at which bacterial cells tend to multiply easily. Trace amounts of Al and S that leached out to the culture medium from APA prepared in concentrated sulfuric acid did not affect the antibacterial activity of APA, indicating that the APA surface exhibits excellent antibacterial activity. It was shown that the surface of APA prepared in concentrated sulfuric acid had excellent culture properties for fibroblasts while exhibiting antibacterial activity. APA prepared in concentrated sulfuric acid can be used in various applications that require excellent antibacterial activity as well as on surfaces that suppress bacterial growth and selectively cultivate target cells.