Olive fruits are seriously deteriorated by pre and postharvest damage due to the attack of insects, such as Bactrocera olaea, which strongly alters the quality of olives. Defence response in olive fruits injured both by pathogens and by mechanical damages has been associated with the enzyme β-glucosidase, which specifically hydrolyses oleuropein, producing highly reactive aldehyde molecules. In situ detection of β-glucosidase activity in olive fruit tissues following injury, which simulates Bactrocera oleae punctures, is reported. The assay was performed in two cultivars showing different degrees of susceptibilities to fly infestation. In both cultivars, the histochemical assay for β-glucosidase showed that within 20 min after the injury, a strong β-glucosidase activity could be observed in the damaged tissues. Thereafter a progressive enzyme inactivation occurred starting from tissues around the boundary of the injury with decrease of the enzyme activity and stopped after 3h. Whereas the mass of active celsreached a distance of (300±50)μm from the edge of the injury. Biochemical analyses showed that in extracts of the injured fruit, β-glucosidase activity rapidly increased within 20 min from injury, thereafter decreasing and reaching values comparable with those in intact fruits. Following puncture, the oleuropein contents did not change significantly in the high susceptibility cultivar, whereas it rapidly decreased in the cultivar showing low susceptibility. The results strongly suggest that olive fruits susceptible towards fly infestation could be related to the ability of the oleuropein-degrading-β-glucosidase to produce the highly reactive molecules in the damaged tissues. As a consequence of puncture, high level of peroxidase activity was detected. This feature also suggested that this enzyme could play a key role in the defence response against insect injuries.