Introduction: Current published reports on the causative agents of acute febrile illness (AFI) in Afghanistan are scarce, and the burden of disease due to flaviviruses is unknown. Methodology: A hospital-based surveillance study for AFI was established in 2008 through 2010 to determine the seroepidemiology of West Nile virus (WNV), tick-borne encephalitis virus (TBEV) and dengue viruses (DENV) using commercial ELISA kits. Due to major logistical challenges, only acute sera were collected. Results: Serological analysis for IgG were as follows: WNV 30.4% (277/913); TBEV 23.4% (214/913); DENV 19.7% (180/913). Single positive IgG reactions for WNV, TBEV and DENV were noted in 11% (100/913), 7.2% (66/913), and 5% (47/913), respectively. Reactivity for all three screened flaviviruses was detected in 44.5% (406/913) of sera. IgM positivity was uncommon, with only 0.5% (5/913), 2.2% (20/913) and 2.6% (8/312) of samples positive for WNV, TBEV, and DENV, respectively. Serological findings were confirmed in random positive samples by neutralization assay. Conclusions: These serological results suggest circulation of WNV, TBEV, and DENV within Afghanistan, with evidence of current or prior infection noted in a significant proportion of patients seeking care for AFI. Obtaining additional information on the prevalence of these and other causes of AFI is paramount for improving the distribution of available limited syndromic treatment and improving the existing health protection policy in Afghanistan.