Two novel influenza A virus-like genomes were detected in fruit bats in Central and South America. However, the biological properties of these bat-derived influenza viruses (BatIVs) are still largely unknown since infectious viral particles have never been isolated from the infected host species. In this study, a reverse genetics approach was used to generate infectious BatIV particles entirely from plasmids encoding full-length sequences in eight gene segments. We inoculated BatIV particles into various cell cultures including bat-derived cell lines and found that BatIVs infected particular bat-derived cells efficiently but not the other cell lines tested. Reassortant viruses between the two BatIVs were also successfully generated and their replication in the susceptible bat cell lines was confirmed. These findings suggest a limited host range and reassortment potential of BatIVs in nature, providing fundamental information for understanding of the ecology of BatIVs.