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Because of its outstanding biological and industrial importance, many efforts have been made to characterize the mycobiota of new environments and their biochemical and biotechnological potentials. Gut mycobiota can be a source of novel yeasts with the potential to be used as probiotics or have industrial applications. In this work, we characterized two as-yet unexplored yeast communities from the intestinal content of the cultured marine Chilean fishes <i>Genypterus</i> <i>chilensis (G. chilensis)</i> and <i>Seriolella violacea</i> (<i>S.</i> <i>violacea</i>). Yeasts were isolated through culture, identified by sequencing their ITS region, and characterized their enzymatic profile with API^®ZYM. <i>Rhodotorula mucilaginosa</i> was identified in both fish species. For the first time, <i>Candida palmioleophila</i>, <i>Candida pseudorugosa</i>, <i>Cystobasidium slooffiae,</i> and a member of the <i>Yamadazyma</i> genus were also identified and described as part of the normal fish gut–microbiota. Furthermore, the diverse enzymatic profile exhibited by some of these isolates suggests that it may be possible to develop novel applications for them, such as new probiotics and other biotechnological applications.