The initial enzymatic degradation behavior of Langmuir monolayer films of a series of biodegradable polyesters at a constant surface pressure was investigated at the air/water interface. The initial degradation of polyester monolayers strongly depended on the structural formula of the polyesters. By the co-polymerization of 3-hydroxyvalerate (3HV) into the poly[(R)-3-hydroxybutyrate] backbone, the critical surface pressure at which the degradation occurs was increased and the rate of enzymatic degradation was retarded because of the inactivity of the enzyme and the hydrophobicity of 3HV. The decrease in the optical purity of poly(l-lactide) (PLLA) delayed the time when the degradation occurred at a constant surface pressure. However, the degradation rate after the critical time was accelerated as the optical purity decreased. These results can be explained by the mutual competition of the preferential degradation of l-lactide units and the density.