West Nile virus (WNV) is a mosquito-borne neurotropic viral pathogen maintained in an enzootic cycle between mosquitoes (vectors) and birds (natural hosts) with equids, humans, and other vertebrates acting as dead-end hosts. WNV activity in Mexico has been reported in several domestic and wild fauna and in humans, and the virus has been isolated from birds, mosquitoes, and humans. However, no serological studies have been conducted in monkeys, and only two in a limited number of crocodiles (Crocodylus moreletii). Here we present data on the prevalence of neutralizing antibodies against WNV in 53 healthy wild monkeys (49 Ateles geoffroyi and four Alouatta pigra), and 80 semi-captive healthy crocodiles (60 C. acutus and 20 C. acutus–C. moreletti hybrids) sampled during 2012. None of the monkey sera neutralized WNV, whereas 55% of the crocodile sera presented neutralizing antibodies against WNV. These results can contribute to the design of surveillance programmes in Mexico.