Foodborne illness constitutes a substantial health and economic burden on society, and most cases are believed to be due to mishandling of food practices. This study aimed to establish the relationship between food handling practices and Campylobacter contamination in commercial restaurant kitchens. The handling practices of chefs during chicken preparation were observed and recorded. Thirty-three chicken samples and 39 environmental samples were collected to detect the presence of Campylobacter. In total, 75 Campylobacter jejuni isolates and 14 Campylobacter coli isolates were identified. All C. jejuni isolates were characterized by whole genome sequencing (WGS) and multilocus sequence typing (MLST). Campylobacter was positive on chickens with a ratio from 50% to 66.67%. In washing and cutting areas, the Campylobacter positive ratio varied from 33.33% to 100%. Hands, boards, and countertop showed the highest loads of bacterial counts. WGS and MLST results indicated that all isolates were grouped into 17 genetic clusters. Isolates belonging to the same sequence type were highly homologous and our findings indicated that C. jejuni was likely transmitted from chicken to other areas within the kitchen. Improper handling practices (i.e., using cloths, washing hands without soap, cleaning boards with knives) caused the surfaces contamination of Campylobacter. Our results indicated that handling practices were a critical point of Campylobacter cross contamination in commercial kitchen premises. Therefore, more attention should be focused on proper handling practices in commercial kitchens to reduce the risk of foodborne illness.