Abstract Background Acute diarrhea is a global health problem, resulting in high morbidity and mortality in children. It has been suggested that enteric pathogen co-infections play an important role in gastroenteritis, but most research efforts have only focused on a small range of species belonging to a few pathogen groups. This study aimed to assess the impact of co-infections with a broad range of enteric pathogens on children aged below five years who suffer from acute diarrhea in southwest China. Method A total of 1020 subjects (850 diarrhea cases and 170 healthy controls) were selected from four sentinel hospitals in Kunming, Yunnan province, southwest China, from June 2014 to July 2015. Stool specimens were collected to detect five virus (rotavirus group A, RVA; norovirus, NoV; Sapovirus, SaV; astrovirus, As; and adenovirus, Ad), seven bacterial (diarrheagenic Escherichia coli , DEC; non-typhoidal Salmonella , NTS; Shigella spp.; Vibrio cholera ; Vibrio parahaemolyticus ; Aeromonas spp.; and Plesiomonas spp.), and three protozoan ( Cryptosporidium spp., Giardia lamblia , and Blastocystis hominis , B. hominis ) species using standard microbiologic and molecular methods. Data were analyzed using the partial least square regression technique and chi-square test. Results At least one enteric pathogen was detected in 46.7 % ( n = 397) of acute gastroenteritis cases and 13.5 % ( n = 23) of healthy controls (χ 2 = 64.4, P