A persistent need exists for effective treatment agents for mycobacterial infections. This research investigated the effectiveness of the Hypericum perforatum herb (commonly known as St John's wort; SJW) in its growth inhibition of mycobacteria. A SJW extract was effective at inhibiting five nonpathogenic Mycobacterium isolates and Bacillus subtilis, but not Escherichia coli. Quantitative studies of concentration sensitivity to the SJW extract were performed with minimal bactericidal concentrations (MBC) ranging from 0.33 to 2.66 mg extract/mL. The SJW compounds hyperforin (Hfn), hypericin (Hpn), and pseudohypericin (Phn) were quantified in the extract using HPLC. The SJW extract solution of 133 mg extract/mL used in this study contained 2.3 mg Hfn/mL, 0.8 mg Hpn/mL, and 2.1 mg Phn/mL. Purified Hfn, Hpn, and Phn were tested for inhibitory activity against Mycobacterium JLS (M. JLS) at similar concentrations used in the crude extract. While Hfn was inhibitory at 46 µg/mL, none of the purified SJW constituents were bactericidal at concentrations corresponding to SJW treatments. Scanning electron microscopy (SEM) analysis of SJW-treated M. JLS cells showed changes in cell surface morphology. Copyright © 2012 John Wiley & Sons, Ltd.