In this study, we compared the antibacterial effectivity of the eggs of six precocial and four altricial bird species using Escherichia coli, based on their eggshell traits. The ultrastructure of eggshell was observed using a scanning electron microscope (SEM). According to SEM results, eggs from precocial birds (chicken, turkey, quail, duck, ostrich, and goose) had cuticle on the eggshells, while eggs from altricial birds (pigeon, budgerigar, munia, and canary) did not. The environment/selection pressure may induce the divergent evolution process in eggs of precocial and altricial birds. The E. coli experiment results showed that chicken, turkey, quail, duck, and goose eggs, with a high cuticle opacity, exhibited a much lower E. coli penetration rate. In contrast, the eggs with poor (ostrich) or without (pigeon, budgerigar, munia, and canary) cuticle exhibited a higher penetration rate. It is suggested that cuticle is a main barrier against bacterial penetration in precocial birds' eggs. Turkey and quail eggs showed the lowest E. coli contamination rate (3.33% and 2.22%, respectively), probably because of the tightly connected nanosphere structure on their cuticle. As for altricial birds' eggs, the eggs of budgerigar, munia, and canary with small pore diameter (0.57 to 1.22 μm) had a lower E. coli penetration rate than pigeon eggs (45.56%, 66.67%, 50%, and 97.78%, respectively, P < 0.05), indicating that pore diameter played a significant role in defending against bacterial trans-shell invasion. We found that eggshell thickness and pore area decreased with egg size. The cuticle quality had no relationship with egg size, but was closely related to the bird species. The E. coli penetration rate of altricial birds' eggs was significantly higher than that of precocial birds' eggs, mainly because the pores are exposed on the eggshell surface and cuticle protection is absent. This study provides detailed information on the eggshell cuticle, which gives insight into the cuticle evolution process that occurred in precocial and altricial bird species. Moreover, the results of E. coli penetration may help understanding the antibacterial behavior in birds.