We detected Neobenedenia girellae infections in 40 species belonging to 12 families of imported marine ornamental fish from a public aquarium in the Mexican state of Yucatan. A total of 348 fish specimens were examined for monogeneans in January 2018 to December 2020. Monogeneans were corroborated morphologically and molecularly with a partial sequence of 28S (region D1–D3) ribosomal DNA and analyzed in a molecular phylogenetic context in combination with other N. girellae sequences available in GenBank. The phylogenetic tree revealed that the specimen found consistently belonged to the N. girellae clade. High infection parameters were detected of N. girellae in most hosts. This identification is relevant to aquarists and aquaculturists in the Gulf of Mexico because N. girellae is considered highly pathogenic in confined fish. This work demonstrates that the importation of ornamental fish coupled with deficient sanitary measures (lack of quarantine areas in distribution centers) contributes to spread of parasites and their establishment within Mexico.