Lizards belonging to the genus Anolis are native to America and have been introduced in many parts of the world. In this work, a gastrointestinal microbiological analysis from Anolis sp. introduced to Tenerife, Canary Island, was carried out. A total of 74 individuals were analyzed by culture and molecular tools. Pseudomonas spp. was the most prevalent bacteria isolated (64.3%), followed by enteropathogenic Escherichia coli with at least one of the investigated virulent genes (stx1, stx2, and eae) (44.6%). The stx2 gene was more prevalent which differs to that reported in other reptiles, probably due to wastewater transmission. Campylobacter spp. was detected in 32.4% of the animals, highlighting the detection of C. jejuni and C. fetus by their relevance to public health. The zoonotic Staphylococcus lugdunensis, found in 14.9% of the animals, was firstly detected in reptiles. Vibrio sp. which is more associated with aquatic environments was found in 10.8% of the lizards in this study, with Vibrio cholerae being found in two of the animals. The prevalence of Salmonella sp. (5.4%) was low, compared with other studies carried out in reptiles. These results indicate that Anolis sp. in Tenerife could be playing a role in the maintenance and spread of the pathogens detected, being a possible risk factor for public health and biodiversity conservation.