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In external fertilizer fish, ovarian fluid (OF) seems to play a key role in fertilization success, improving spermatozoa swimming performance. These OF/sperm interaction mechanisms are frequently species-specific and/or population-specific and could decrease the risk of genetic introgression of wild populations from introduced or escaped zootechnical individuals. The Mediterranean brown trout (<i>Salmo cettii</i>) is threatened by genetic introgression with strains of domestic brown trout (<i>Salmo trutta</i>) that were introduced for recreational purposes. The aim of our study was to test if native <i>S. cettii</i> females, rather than zootechnical S. <i>trutta</i>, produce OF with a greater ability to upregulate the sperm motility of conspecific males. Thus, we compared the sperm swimming performances of males inhabiting the Biferno River (Molise region—Southern Italy) activated in native <i>S. cettii</i> vs. zootechnical <i>S. trutta</i> female’s OFs. In our study, native females’ OFs (20% diluted), compared to spring water, has the ability to significantly boost the sperm performance of the autochthonous males, while zootechnical <i>S. trutta</i> fails. These preliminary results suggest that OF-sperm interactions could potentially influence or direct the hybridization mechanisms involving the native Mediterranean trout inhabiting the Biferno River and the domestic lineage of brown trout introduced in the past.