Microcotyle is one of the most diverse and controversial genera within the family Microcotylidae. To date, 131 species have been described in Microcotyle; however, more than half have been transferred to other genera, and several others have poor descriptions. Therefore, less than half of all Microcotyle species may be considered valid. In Chile, two species have been recognized, and unidentified Microcotyle have been found on several littoral fish, but there has been no effort to properly identify them. In this study, two new species of Microcotyle are taxonomically described from intertidal fish of the central (33°S) and south-central (36°S) regions of Chile. In this study, Microcotyle sprostonae n. sp. (collected mainly from Scartichthys viridis in central Chile) and M. chilensis n. sp. (collected mainly from Calliclinus geniguttatus in south-central Chile) were identified based on morphological and molecular analyses (ITS2 and 18S genes). Both species of Microcotyle principally differed from one another and from other valid species in the number of testes and clamps. The two new species also differed from one another by one base pair in the ITS2 and 18S genes and differed from other species of Microcotyle by several base pairs of both genes. Intertidal fish are mostly endemic to the Pacific coast of South America, and they have a limited geographical distribution that does not overlap with the type hosts of other Microcotyle species. Therefore, the two new species described here are distinguished from other congeneric species by morphological, genetic, and biological characteristics.