The objective of this study was to examine antibacterial, antifungal, and antimycobacterial properties of flex aquifolium L. (Aquifoliaceae) growing in Turkey. The ethanol, ethyl acetate, chloroform, and n-hexane extracts prepared from the leaves of I. aquifolium were tested against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterobacter aerogenes, Proteus vulgaris, Salmonella typhimurium, and Candida albicans for antibacterial and antifungal evaluation using the microdilution broth susceptibility assay. In addition, antimycobacterial activity of the crude extracts of I. aquifolium was evaluated by microplate Alamar blue assay. The results showed that the extracts tested, except n-hexane, possessed moderate antibacterial and antifungal activity varying from 62.5 to 250 mu g/mL. On the other hand, the ethanol extract of the leaves exhibited a minimum inhibitory concentration value (MIC) against Mycobacterium tuberculosis H37Ra strain of 200 mu g/mL.