In this study, two experiments were performed to investigate the anesthetic efficiency, hematological and biochemical effects of thymol on Nile tilapia, Oreochromis niloticus. In the first experiment, in order to estimate thymol anesthetic efficacy, tilapia juveniles were exposed to anesthetic baths at six different concentrations of thymol (10, 20, 40, 60, 80, and 100 mg L−1) and the induction and recovery times were recorded. The second experiment was performed with five treatments: CON (non-anesthetized fish), TH1 (anesthesia with 80 mg L−1 thymol within 1 min), TH5 (anesthesia with 30 mg L−1 thymol within 5 min), EU1 (anesthesia with 80 mg L−1 eugenol within 1 min), and EU5 (anesthesia with 30 mg L−1 eugenol within 5 min). The results showed that thymol at 10 mg L−1 concentration failed to induce anesthesia; however 20–100 mg L−1 concentrations induced anesthesia within 491–56.3 s. A strong negative relationship was detected between thymol concentrations and induction times (R2 = 0.906). Anesthesia had no significant effects on plasma albumin, triglyceride, cholesterol, alanine aminotransferase, and the blood mean corpuscular hemoglobin levels. The anesthetized fish exhibited significant elevations in plasma cortisol, glucose, lactate, and the blood hematocrit and mean corpuscular volume; these parameters exhibited significant elevations in the fish anesthetized by 30 mg L−1 anesthetics, compared to the 80 mg L−1. Plasma globulin, total protein, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, catalase, superoxide dismutase, and the blood hemoglobin and red blood cell significantly increased, as mean corpuscular hemoglobin concentration decreased, in the fish anesthetized by 30 mg L−1 anesthetics, compared to the CON fish. The anesthetized fish showed similar plasma glutathione levels, all significantly lower than the CON fish. The EU5 fish exhibited significant elevation in the plasma malondialdehyde levels, compared to the CON fish. In conclusion, thymol anesthetic efficacy was similar to eugenol in Nile tilapia weighing ~40 g at water temperature of 26 °C. Considering the physiological responses, slight but significant elevations in plasma proteins, lactate, lactate dehydrogenase, and malondialdehyde levels attest that thymol may induce less stress, hypoxia, and oxidative stress in the fish than eugenol, but further studies are needed to provide robust and clinically applicable data to support this hypothesis.