Brazoran virus was first isolated from Culex mosquitoes in Texas in 2012, yet little is known about this virus. We report the isolation of this virus from Culex erraticus from southern Florida during 2016. The Florida strain had a nucleotide identity of 96.3% (S segment), 99.1% (M segment), and 95.8% (L segment) to the Texas isolate. Culex quinquefasciatus and Aedes aegypti colonies were subsequently fed virus blood meals to determine their vector competence for Brazoran virus. Culex quinquefasciatus was susceptible to midgut infection, but few mosquitoes developed disseminated infections. Aedes aegypti supported disseminated infection, but virus transmission could not be demonstrated. Suckling mice became infected by intradermal inoculation without visible disease signs. The virus was detected in multiple mouse tissues but rarely infected the brain. This study documents the first isolation of Brazoran virus outside of Texas. Although this virus infected Ae. aegypti and Cx. quinquefasciatus in laboratory trials, their vector competence could not be demonstrated, suggesting they are unlikely vectors of Brazoran virus.