Etiological carriers and the excretion of the pathogens causing hand, foot, and mouth disease (HFMD) in healthy persons, patients, and asymptomatic persons infected with HFMD as ongoing infection sources may play an important role in perpetuating and spreading epidemics of HFMD. The aims of this study were to determine the carrier status of EV-A71 and CV-A16 in healthy populations, as well as the duration of EV-A71 and CV-A16 shedding in the stools of HFMD patients in an epidemic area of southwest China. A cross-sectional study and a follow-up study were conducted in three HFMD endemic counties of Yunnan Province. Six hundred sixty-seven healthy subjects were recruited to participate in the cross-sectional study, and two stool specimens were collected from each subject. Among the healthy subjects, 90 (13.5%) tested positive for viral isolation, but neither EV-A71 nor CV-A16 was detected in healthy individuals. Of the 150 patients with probable HFMD, 55.3% (83/150) tested positive for viral isolation with presented serotypes such as EV-A71 (51.81%, 43/83), CV-A16 (32.53%, 27/83), other EVs (13.25%, 11/83), and mixed EV-A71 and CV-A16 (2.41%, 2/83). The longest duration of EV-A71 and CV-A16 shedding in stool specimens from patients with HFMD was >46 days after onset. The positive rate of EV-A71 in the stool specimens of confirmed patients dropped to 50% by the end of the third week, and the same occurred with CV-A16 by the end of approximately the seventh week after onset. Although carriers of major causative agents of HFMD in healthy populations are fewer in number, the prolonged shedding of pathogens in patients with HFMD may serve as an important factor in perpetuating and spreading HFMD epidemics.