The objective of this study was to assess the influence of a Valeriana officinalis extract on the activity of the drug-metabolising enzymes cytochrome P450 2D6 (CYP2D6) and 3A4. Probe drugs dextromethorphan (30 mg; CYP2D6 activity) and alprazolam (2 mg; CYP3A4 activity) were administered orally to healthy volunteers (n = 12) at baseline and again after exposure to two 500-mg V. officinalis tablets (1000 mg) nightly for 14 days. The V. officinalis supplement contained a total valerenic acid content of 5.51 mg/tablet. Dextromethorphan to dextorphan metabolic ratios (DMRs) and alprazolam pharmacokinetics were determined at baseline and after V. officinalis treatment. The DMR was 0.214 ± 0.025 at baseline and 0.254 ± 0.026 after valerian supplementation (P > 0.05). For alprazolam, the maximum concentration in plasma was significantly increased after treatment with V. officinalis (25 ± 7 ng/ml vs. 31 ± 8 ng/ml; P < 0.05). There were no significant differences in other pharmacokinetic parameters at baseline and after V. officinalis exposure (all P values = 0.05; time to reach maximum concentration in plasma, 3.0 ± 3.2 vs. 3.1 ± 2.1 h; area under the plasma concentration vs. time curve, 471 ± 183 vs. 539 ± 240 h × ng × ml⁻¹; half-life of elimination, 13.5 ± 4.3 vs. 12.2 ± 5.6 h). Our results indicate that although a modest increase was observed in the alprazolam Cmax, typical doses of V. officinalis are unlikely to produce clinically significant effects on the disposition of medications dependent on the CYP2D6 or CYP3A4 pathways of metabolism.