Toxicity of nootkatone was determined in laboratory assays against unfed nymphs of Amblyomma americanum L., Dermacentor variabilis (Say), Ixodes scapularis Say, and Rhipicephalus sanguineus Latreille. We determined the 50% lethal concentration (LC₅₀) and 90% lethal concentration (LC₉₀) of nootkatone by recording tick mortality 24 h after exposure in treated glass vials. Nymphs were susceptible to nootkatone with LC₅₀ values of 0.352, 0.233, 0.169, and 0.197 µg/cm², and LC₉₀ values of 1.001, 0.644, 0.549, and 0.485 µg/cm² for A. americanum, D. variabilis, I. scapularis, and R. sanguineus, respectively. The LC₅₀ value for R. sanquineus was not significantly different from D. variabilis or I. scapularis. Other LC₅₀ comparisons were significantly different. The LC₉₀ for A. americanum was higher when compared with the three other tick species, which were not significantly different. Because nootkatone is volatile, we measured the amount of nootkatone recovered from duplicate-treated vials before tick exposure and from vials after tick exposure. Nootkatone recovered from vials before exposure ranged from 82 to 112% of the expected amounts. The nootkatone recovered after the 24-h exposure period ranged from 89% from vials coated with higher concentrations of nootkatone, down to 29% from vials coated with low nootkatone concentrations. Determination of the nootkatone residue after vial coating demonstrated loss of the active compound while verifying the levels of tick exposure. Toxicity of low concentrations of nootkatone to the active questing stage of ticks reported in this study provides a reference point for future formulation research to exploit nootkatone as a safe and environment-friendly tick control.