Trophic conditions and water temperature strongly influence bivalve physiological processes and metabolism. In black-lip pearl oyster Pinctada margaritifera, these parameters have been shown to affect shell biomineralization. The present study investigated the effect of preoperative food level (i.e., microalgal concentration) and temperature on pearl biomineralization. Donor and recipient oysters were conditioned at different levels of food and temperature during the preoperative phase to evaluate the influence of these factors on 1) pearl retention rate (grafting success), 2) expression of genes involved in biomineralization in the mantle and pearl sac and 3) pearl quality traits. Our study confirmed the influence of both microalgal concentration and temperature on shell growth. Food level of donor oysters was decisive for pearl biomineralization, with donors that had been fed at a high microalgal concentration producing pearl sacs with significantly higher biomineralization capabilities and faster nacre establishment during early stages of pearl formation. However, food level showed no effects on quality traits of the pearls harvested 12 months postgrafting, while preoperative temperature only influenced the relative expression of two genes in pearl sacs at 12 months postgrafting. No significant effects of the preoperative conditioning of recipient oysters were detected in either experiment considering gene expression measurements and pearl quality traits. However, mortality was significantly lower in grafted recipient oysters fed at an intermediate trophic level. Finally, pearl weight was shown to be positively correlated with recipient oyster growth.