The appearance and spread of antimicrobial resistance (AMR) in bacteria in natural environments and wildlife are related to agricultural and livestock activities and are a global health and conservation problem. We assessed the presence of AMR genes in Escherichia coli isolated from black howler monkeys (Alouatta pigra), sheep (Ovis aries), cattle (Bos taurus), and horses (Equus caballus) from a highly fragmented forest in southern Mexico. Fresh fecal samples were collected using swabs, seeded on eosin–methylene blue agar, and E. coli colonies identified by PCR; multiplex-PCR was performed on E. coli DNA for the detection of 10 AMR genes from four families (sulfonamides, tetracycline, β-lactamase, and chloramphenicol). We detected E. coli in 94% (48/51) of fecal samples, of which 33% (16/48) tested positive for at least one AMR gene. We detected AMR genes in at least one individual from each sampled animal species, with the most prevalent genes being tet(B) 18% (9/48), sul2 14% (7/48), sul1, and blaTEM 12% (6/48). Sheep samples contained AMR genes from the four families of antibiotics detected in this study and 50% (5/10) tested positive for the presence of at least one gene. A total of 12% (2/16) of fecal samples from black howler monkeys tested positive for AMR genes. The presence of AMR genes in A. pigra and domestic animals has not been reported in the Balancán area of Tabasco, Mexico. Transmission of AMR bacteria from domestic animals to monkeys is rare; however, this is a potential health risk for wildlife and species conservation.