The induction of plant resistance against pests is considered a potential method of controlling mite infestation as it restricts the use of chemical pesticides in herbal crops. Our goal was to investigate whether plant physiological response to mite feeding varied depending on basil cultivar and/or duration of mite infestation. The effect of plant acceptance, mite mortality rate, and changes in physiological parameters: malondialdehyde content (MDA), hydrogen peroxide (H2O2) concentration, and antioxidant enzyme activities, including guaiacol peroxidase (GPX) and catalase (CAT) were examined in this study. Tetranychus urticae Koch (Acari: Tetranychidae) infestation induced oxidative stress in three Ocimum basilicum L. cultivars: ‘Sweet basil,’ ‘Purpurascens,’ and ‘Fino Verde.’ The analysis of mite behavior and alteration in metabolic plant profiles showed different sensitivities of basil cultivars to biotic stress that were dependent on the cultivar and duration of infestation. All basil plants were suitable as host plants for T. urticae, but they varied in the level of susceptibility to mite feeding. O. basilicum ‘Fino Verde’ was the most suitable host for the twospotted spider mite. In turn, O. basilicum ‘Purpurascens’ was characterized by the lowest level of susceptibility to T. urticae feeding. The lowest acceptance, the highest mortality of twospotted spider mite individuals as well as decreased levels of H2O2 and MDA, significantly increased GPX activity and low level of CAT activity were recorded in O. basilicum ‘Purpurascens’ leaves. Research on plant responses to biotic stress can inform breeding cultivars resistant to arthropod attack.