For better understanding of the antistaling effect of starch-hydrolyzing enzymes, maltose-, maltotriose-, or maltotetraose-producing enzymes were applied to bread mix and the retrogradation rate of the bread was determined using differential scanning calorimetry. A new amylase isolated from Bacillus subtilis SUH 4-2, which selectively produces maltose and maltotriose from starch solution (amylase II), and another amylase from Streptomyces albus KSM-35, mainly producing maltotetraose and maltotriose (amylase IV), were cloned, characterized, and evaluated as antistaling agents for bread. Addition of amylase II or amylase IV significantly reduced the bread staling rate during 7 days of storage (p < 0.05), and especially amylase IV was as effective as a commercial enzyme, Novamyl. Analyses of the maltooligosaccharide composition of bread suggest that maltotriose and maltotetraose produced by the enzyme reaction are responsible for retarding bread retrogradation.