Objectives: This study was performed to investigate hygienic behavior of food handlers and general population focusing on awareness of hand-washing and the microbial load of their hands. Methods: A questionnaire survey and microbiological analysis were carried out for sixty-four people each. Samples for microbiological analysis were collected through the glove-juice method from the hands, and were analyzed for the presence of aerobic plate counts, total coliform, fecal coliform, Escherichia coli, Staphylococcus aureus, and Salmonella spp. according to the Food Code of Korea. Results: In the survey, significant differences between the food handlers and general population (p < 0.01) were found in hand-washing frequency, duration, use of hand-washing agents, washing parts of hands, hand-drying method, and method of turning off water. In eight different situations among the ten particular situations in their daily life, more food handlers responded to wash their hands than general population (p < 0.05). Bacterial load on hands with general population was consistently higher than with food handlers (p < 0.05), however, percentages of positive hands of S. aureus and Salmonella spp. were not. Conclusions: Poor hand hygiene practices were indicated by the positive results for E. coli, S. aureus, and Salmonella spp. on the hands of some respondents in both groups. This study reveals that there is the need for programs or campaigns to increase hand-washing practices of both groups.