Inadequate food handling and poor hand hygiene play a major role in the occurrence of foodborne diseases. The objective of this study was to find out if the level of microbial contamination on the hands of food preparers varies by time during their working period. This study focused on the contamination of aerobic plate count,total and fecal coliforms, Escherichia coli, Staphylococcus aureus, and Salmonella spp. Sampling from left hand and right hand of twenty food preparers was done with glove-juice method at every two hours during their work. Microbiological testing was conducted according to the Food Code of Korea. The microbiological load on the hands was changed over time. Samples taken from their hands before work and at 8 hours showed higher levels of bacteria than those taken at 2, 4, and 6 hours during work and/or after work (p < 0.05). The contamination levels of microorganisms were consistently higher in right hand than in left hand. Poor hand hygiene practices were indicated by the positive results for total and fecal coliforms, E. coli, S. aureus, and Salmonella spp. on the hands of some food preparers. This study indicates food preparers' hands can be a vehicle of pathogen during their work. The results of this study emphasize the importance of hand hygiene education and training targeting the food preparers.