The objective of the study was to investigate the occurrence of H. pullorum in chicken hens’ eggs and to determine their in vitro susceptibility to various antimicrobial agents. A total of 300 fresh commercial chicken eggs of Balady native breed and poultry farms (PF) were collected from both Assiut and Qena Governorates in Egypt. Every five eggs represented one sample (mixed contents of five eggs). The eggshell and eggshell contents were examined for the presence of Helicobacter species by conventional methods, next confirmed with PCR. The highest incidence of contamination with Helicobacter species was recorded in eggs from Assiut Governorate poultry farms. The obtained results of Helicobacter pullorum (H. pullorum) isolation revealed that poultry farms egg shells and contents were less contaminated in both Governorates than Balady ones. Additionally, fresh egg contents of poultry farms in both Governorates were free from H. pullorum. All 12 isolates that were conventionally classified as carrying H. pullorum occurred free from the organism when tested with PCR method by detection of one PCR product on agarose gel that matched the predicted size of 477 bp that corresponding to 16S rRNA region of the gene. H. pullorum show in vitro susceptibility to almost all tested types of antibiotics except for ampicillin, ceftriaxone, and sulphamethoxazole trimethoprim.