Background: Understanding enzootic cycles is key to arbovirus control. Rodents are primary reservoirs of Venezuelan equine encephalitis virus (VEEV), whereas the vertebrate host of Madariaga virus (MADV) remains unknown. Culex (Melanoconion) mosquitoes are considered primary vectors. We studied small mammals and mosquito ecology in the Darién province and Panamá Oeste province, two enzootic regions of Panama. Additional alphavirus seroprevalence was assessed in small mammals. Methods: Small mammals were trapped using Sherman and Tomahawk traps, and blood samples were tested for MADV and VEEV neutralizing antibodies using plaque reduction neutralization test and attempt viral isolation and generic alphavirus RT-PCR from small mammal tissues. Mosquitoes were collected concurrently using Centers for Disease Control and Prevention (CDC) light, Trinidad, and resting traps. Diversity and similarity were analyzed using standard ecological indices. Results: We sampled 599 small mammals (16 species). MADV seroprevalence was highest in Los Pavitos (9.0%) and restricted to Darién; VEEV was more widespread, peaking in El Cacao (27.3%) and El Real (20.4%). Oryzomys couesi and Transandinomys bolivaris showed elevated MADV seroprevalence; T. bolivaris and Proechimys semispinosus had higher VEEV seroprevalence. Among 4118 mosquitoes, Coquillettidia venezuelensis was most abundant. El Real had the highest species richness and diversity. Geographic proximity influenced community similarity. Conclusions: Spatial differences in host and vector communities may reflect distinct transmission patterns of MADV and VEE. These findings could contribute to better understanding of alphavirus ecology and potentially inform public health strategies in endemic regions.