The two-spotted spider mite (Tetranychus urticae Koch) is a major agricultural pest, with increasing resistance to synthetic pesticides thereby, driving the search for natural alternatives. This study evaluated the acaricidal and antioxidant activities of tea tree oil (TTO, Melaleuca alternifolia (Maiden and Betch) Cheel), pumpkin seed oil (PSO, Cucurbita pepo L.), and wheat germ oil (WGO, Triticum aestivum L.) against T. urticae. Laboratory trials determined LC50 and LC90 values for adults and eggs. Biochemical effects on mites surviving TTO exposure were assessed by analyzing Glutathione S-transferase (GST), Acetylcholinesterase (AchE), Carboxylesterase (CarE), and α-esterases activities. Antioxidant activity was evaluated via DPPH and ABTS assays. Gas Chromatography-Mass Spectrometry (GC-MS) identified 24 compounds in TTO, with 4-Terpineol (36.65%) and γ-Terpinene (17.66%) as major components. TTO showed the highest acaricidal activity (LC50 of 0.3% for adults and 1.8% for eggs), outperforming PSO and WGO. TTO exposure significantly disrupted key enzymatic activities, impairing mite survival. Among the oils, TTO exhibited the strongest antioxidant activity. The antioxidant assays revealed that while all three oils demonstrated dose-dependent antioxidant effects, TTO was markedly more effective than PSO and WGO, although less potent than vitamin C and Trolox. Additionally, TTO exposure resulted in significant reductions in detoxification enzyme activity, particularly GST and AchE, highlighting a biochemical mechanism underlying its acaricidal action. The lipophilic properties of TTO likely enhance its penetration through the mite cuticle, increasing its efficacy. These findings support the use of plant-derived oils as eco-friendly alternatives for sustainable pest management and suggest potential for further development into natural pesticide formulations.