In this study, we examined the virus diversity in serum samples from Nicaraguan children with unknown acute febrile illness. We performed Virochip microarray and deep sequencing analyses on 7 positive control and 123 undiagnosed samples. Both of these methods succeeded in detecting the expected virus in the positive control samples. Virochip analysis produced putative viral hits in 10/123 (8%) of the previously negative samples, whereas deep sequencing revealed virus or virus-like sequences in 45/123 (37%). This study demonstrates the utility of these metagenomic strategies to detect virus sequence in multiple human serum samples and is the first to utilize second-generation sequencing to simultaneously investigate many cases of acute unknown tropical illness.