Diarrhea is a daily public health song in developing countries like Tanzania. The causative agents are theoretically known almost to everybody. However, the eradication of this killer disease for the under-fives is an enigma. This study aimed to provide update advantages of molecular diagnostic versus conventional methods as regards to acute diarrhea, and to determine bacterial causes of diarrhea among children aging five years and below in Dar Es Salaam, Tanzania, using multiplex PCR technique. Samples were collected from the under-fives from district hospitals in Dar Es Salaam city between June 2010 and February 2014. This included children admitted due to acute and/ or chronic diarrhea. A total of 3600 stool samples were analyzed, of which 1800 samples were from diarrhea cases and 1800 samples from normal control cases. About 1080 (60%) of the patients recruited were aged less than 3 years and 983 (54.6%) were males. Diarrheagenic bacteria were isolated and identified using conventional stool cultures then were characterized by mPCR. Pathogenic bacteria were detected in 67.7% of the cases and in 20% of the controls. The pathogenic bacteria most strongly associated with diarrhea disease were diarrheagenic Escherichia coli (21.6% of cases, 6% of controls), Shigella spp. (16.1% of cases, 5% of controls) and Salmonellae, (10.6% of cases, 3% of controls. The pathogenic bacteria were mostly from children aging from 24 months and above. Diarrheagenic bacteria play an important role in relation to childhood diarrhea aging from two years and above. Proper diagnostic methods, prevention and control through fostering good hygiene and sanitation to water and food should be emphasized especially to oral-faecal age.