Enteroaggregative Escherichia coli (EAEC) is an emerging pathogen frequently associated with acute diarrhea in children and travelers to endemic regions. EAEC was found the most prevalent bacterial diarrheal pathogen from hospitalized Bolivian children less than five years of age with acute diarrhea from 2007 to 2010. Here, we further characterized the epidemiology of EAEC infection, virulence genes, and antimicrobial susceptibility of EAEC isolated from 414 diarrheal and 74 non-diarrheal cases. EAEC isolates were collected and subjected to a PCR-based virulence gene screening of seven virulence genes and a phenotypic resistance test to nine different antimicrobials. Our results showed that atypical EAEC (a-EAEC, AggR-negative) was significantly associated with diarrhea (OR, 1.62, 95% CI, 1.25 to 2.09, p < 0.001) in contrast to typical EAEC (t-EAEC, AggR-positive). EAEC infection was most prevalent among children between 7–12 months of age. The number of cases exhibited a biannual cycle with a major peak during the transition from warm to cold (April–June). Both typical and a-EAEC infections were graded as equally severe; however, t-EAEC harbored more virulence genes. aap, irp2 and pic were the most prevalent genes. Surprisingly, we detected 60% and 52.6% of multidrug resistance (MDR) EAEC among diarrheal and non-diarrheal cases. Resistance to ampicillin, sulfonamides, and tetracyclines was most common, being the corresponding antibiotics, the ones that are frequently used in Bolivia. Our work is the first study that provides comprehensive information on the high heterogenicity of virulence genes in t-EAEC and a- EAEC and the large prevalence of MDR EAEC in Bolivia.