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Intestinal diarrheagenic polyparasitic infections are among the major public health concerns in developing countries. Here we examined stool specimens by microscopy, DNA dot blot and polymerase chain reaction (PCR) to evaluate the co-infection of four principal protozoans among amoebic dysentery cases from Northeast Indian population. The multiplex PCR confirmed Entamoeba histolytica (8·1%), Entamoeba dispar (4·8%) and mixed infection of both the parasites (3·4%) in 68 of 356 stool specimens that were positive in microscopy and/or HMe probe based DNA dot blot screening. The prevailing parasite that co-exists with E. histolytica was Giardia duodenalis (34·1%), followed by Enterocytozoon bieneusi (22·0%), Cryptosporidium parvum (14·6%) and Cyclospora cayetanensis (7·3%, P = 0·017). Symptomatic participants (odds ratio (OR) = 4·07; 95% confidence interval (CI) = 1·06, 15·68; P = 0·041), monsoon season (OR = 7·47; 95% CI = 1·40, 39·84; P = 0·046) and participants with family history of parasitic infection (OR = 4·50; 95% CI = 1·16, 17·51; P = 0·030) have significant association with overall co-infection rate. According to molecular consensus, comprehensive microscopy yielded 3·4% (12/356) false-negative and 7·6% (27/356) false-positive outcome, suggesting an improved broad-spectrum PCR-based diagnostic is required to scale down the poor sensitivity and specificity as well as implementation of integrated control strategy. [ABSTRACT FROM PUBLISHER]