This study investigated the prevalence of 19 virulence factors and biofilm production in 86 EAEC isolates causing diarrhea in children less than 5 years of age from Ifakara, Tanzania. Virulence factors were detected by PCR, whereas biofilm production was determined using a microtiter plate assay. No virulence factor, with the exception of the aat gene used to identify EAEC, was detected in 11/86 isolates (12.8%). The most frequently detected virulence factor was the aggR gene in 53 (61.6%) EAEC, followed by antigen 43 in 33.7%, dispersin in 26.7%, yersiniabactin in 22.1%; autrotransporter Sat in 20.9%; Shigella enterotoxin-1 in 16.3%, and heat-stable toxin-1 in 15.1%. Biofilm was produced in 66/86 (76%) isolates. AggR was the most prevalent virulence factor in the biofilm-forming group (65% versus 38%, P = 0.032). These results again show the high heterogeneity of virulence factors among EAEC isolates causing diarrhea in children, and that biofilm may be an important virulence factor, strongly associated with the presence of AggR.