Background: Diarrheal disease remains a leading cause of illness and death, particularly in low-income countries. Its burden, microbiological causes and risk factors were examined in children aged 0-59 months living in Manhiça, rural southern Mozambique. Methods: Trends of diarrhea-related burden of disease were estimated during the period 2001-2012. A prospective, age-stratified and matched (by age, gender and geographical origin), case-control study was conducted during 2007-2011. Clinical, epidemiology, anthropometric measurement and fecal samples obtained from recruited children were used to estimate moderate-to-severe diarrhea (MSD) weighted attributable fractions. Results: Over the last decade the incidence of acute diarrhea has dropped by about 80%. Incidence of MSD per 100 child years at risk for the period 2007-2011 was 9.85, 7.73 and 2.10 for children aged 0-11, 12-23 and 24-59 months respectively. By adjusted population attributable fractions, most cases of MSD were due to rotavirus, Cryptosporidium, ETEC ST (ST only or ST/LT), Shigella and Adenovirus 40/41. Washing hands and having facilities to dispose child's stools were associated with a reduced risk of MSD, while giving stored water to the child was associated with an increased risk of MSD. Conclusions: Despite the predominantly decreasing trends observed throughout the last decade, diarrheal diseases remain today a major cause of morbidity among children aged 0-59 months living in this rural Mozambican area. Rotavirus, cryptosporidium, Shigella, ETEC ST and Adenovirus 40/41 were the most important aetiologies of MSD. Thus, well-known preventive strategies such as washing hands, improving the treatment of stored water, having facilities to dispose children stools, and accelerating the introduction of the rotavirus vaccine should be promoted on a wider scale to reduce the current burden of diarrheal diseases.