Bird eggs are in contact with intestinal microbiota at or after oviposition, but are protected from bacterial translocation by a glycoprotein cuticle layer, the shell, and internal membranes. In a preliminary study, turkey eggs were hatched in a germ-free environment. Firmicutes 16S rRNA gene was detected in the cecal microbiota of hatched poults, suggesting that poults may acquire spore-formers by exposure to shell contents during hatching. Generating gnotobiotic poults for research requires elimination of bacteria from the egg's surface without damaging the developing embryo. The ability of different disinfectants and antiseptics to eliminate eggshell bacteria without harming the developing embryo was tested. Different classes of disinfectants and antiseptics (halogens, biguanidines, and oxidants) were selected to target spores and vegetative bacteria likely present on the egg's surface. Eggs were treated by fully immersing in heated antiseptic (betadine or chlorhexidine) or disinfectant (alkaline bleach, acidified bleach, chlorine dioxide, Oxysept-333, or Virkon S) solutions for up to 15 minutes. Shells were aseptically harvested for aerobic and anaerobic culturing of bacteria. Toxicity to the developing embryo was assessed by gross evaluation of developmental changes in treated eggs incubated up to 27 d of embryonation. Halogen disinfectants acidified bleach and chlorine dioxide, and oxidants Oxysept-333 and Virkon-S eliminated viable bacteria from eggshells. However, addition of oxidants, alone or in combination with other treatments, produced significant (P < 0.05) embryotoxicity. The combination treatment of acidified bleach, chlorine dioxide, and betadine produced minimal embryotoxicity and eliminated viable bacteria from whole turkey eggs, and produced hatched poults in a gnotobiotic isolator. As a control, eggs were treated with PBS, incubated, and hatched under germ-replete conditions. After hatching, poults were euthanized and treated poults had no detectable bacterial growth or 16S rRNA gene qPCR amplification, demonstrating that acidified sodium hypochlorite, chlorine dioxide, and betadine safely hatched gnotobiotic poults. Generation of germ-free poults is an important tool and will be used to evaluate the host-pathogen interaction by foodborne pathogens such as Campylobacter spp.