In olive micropropagation, Copper (Cu) promotes metabolic activity at optimal levels but exerts toxic effects and induces stress and cellular damage when present at excessive concentrations. The present study examined in vitro olive (cv. Moraiolo) shoot cultures under varying Cu concentrations to evaluate the impact of Cu-induced stress on shoot growth and development, as well as the associated physiological and biochemical tolerance mechanisms. Olive shoots were cultured on OM medium (as a control) supplemented with 50, 100, 200, or 300 µM CuSO₄·5H₂O. Morphological and biochemical analyses showed that up to 50 µM Cu did not cause visible stress symptoms or impair growth, while higher concentrations (100–300 µM) significantly suppressed or inhibited vegetative growth, and caused a marked reduction in photosynthetic pigments. The contents of oxidative stress markers, hydrogen peroxide and malondialdehyde, increased with rising Cu concentrations, serving as reliable indicators of severe stress conditions. Non-enzymatic antioxidants, glutathione, ascorbic acid and proline, increased with higher Cu concentrations, playing a protective role against oxidative damage. These findings provide insight into the tolerance mechanisms of olive shoots under Cu stress, offering useful information for optimizing in vitro micropropagation and understanding Cu toxicity in plant tissue culture.