A neutralizing antibody (NTAb) survey on UNA and Mayaro viruses in black howler monkeys (Aloutta caraya) from subtropical regions of Argentina and Paraguay was carried out. Risk factors for infection in monkeys were analyzed. No positive sera for Mayaro virus were detected. A prevalence of 73% (61/84) of NTAb against UNAV was detected with titers ranging from 20 to 1280. According to the statistical analysis performed, the monkey's age was a significant risk factor, but not the origin or sex. This is the first report of Alouatta caraya infection by UNAV and the first record of its activity in Paraguay.