BACKGROUND: Blueberry production has generated great commercial expectations, therefore for its agricultural expansion it is necessary to overcome the challenges at the time of mass propagation. OBJECTIVE: Evaluate the effect of a set of factors influencing micropropagation, as well as the influence of substrates on the ex vitro morpho-physiological performance of blueberry seedlings. METHODS: A set of protocols were developed to optimize all stages of micropropagation (aseptic establishment, multiplication, rooting, and acclimatization) of blueberries. RESULTS: Explants immersed in 1.5% NaClO for 8 min and then in 0.1% HgCl2 for 2 min achieved 100% sterility and a viability rate of 86.67% for 'Biloxi' and 93.33% for 'Bluecrop'. At the multiplication stage, the maximum number of shoots of 'Biloxi' (3.53) and 'Bluecrop' (2.27) were obtained on the medium supplemented with 0.2 and 10 mg L-1 silver nanoparticles (AgNPs), respectively. The percentage of in vitro rooting was significantly improved on media containing activated charcoal, with levels between 80% and 100%. In the acclimatization phase, plants grown in a substrate composed of peat and cocomix® (2:1 ratio) showed greater uniformity and better morpho-physiological behavior. CONCLUSIONS: The present results could be successfully used for large-scale commercial production of blueberries of the varieties 'Biloxi' and 'Bluecrop'.