Fruit crops of the genus Chaenomeles Lindl are considered today as a superfood due to accumulation of biologically active compounds with antioxidant ability and known health-promoting properties. Successful introduction of this non-traditional culture in the steppe zone of Ukraine characterised by an unfavourable climate suggests the functioning of effective protective mechanisms in plants, including those that can be provided by the influence of endophytic microorganisms. However, there is little information about the endophytic community of Chaenomeles plants. Herein, the current study was aimed to isolate the endophytic fungi from the Ch. speciosa fruits and evaluate their biological activities against the phytopathogens. The study was carried out based on the collection of the Botanical Garden of Oles Honchar Dnipro National University (Dnipro city, Ukraine). Three media, namely PDA, MPA, and Gause’s medium were used for isolation of endophytic fungi. Colonies of isolates for identification were grown on PDA, Czapek's agar, and Czapek’s yeast autolysate media. Six fungal endophytic isolates derived from both peel and pulp of Ch. speciosa fruits have been morphologically identified using macroscopic and microscopic techniques, and assigned to the genus Penicillium (sections Chrysogena, Penicillium, Viridicata), and genus Talaromyces (section Talaromyces). Species P. expansum, P. viridicatum, and P. hirsutum were identified among the peel isolates, while P. chrysogenum, P. cyclopium, and P. purpurogenum were among the pulp isolates. Antagonistic ability of the endophytic isolates against phytopathogenic fungi was evaluated using the dual culture method. The results showed moderate to high antifungal capacity of the endophytic isolates against the phytopathogenic strains of the Fusarium genus. The growth inhibition of F. culmorum mycelium due to the influence of endophytic isolates was 51.5–81.3%, and the inhibition of the growth of F. oxysporum colonies was in the range of 68.4–86.6% as compared with control. There were no significant differences in the antagonistic ability between endophytic isolates derived from the peel and pulp of the fruit. Taken together, our findings indicated the great potential of the endophytic fungi from Ch. speciosa fruits as a source for the development of biocontrol agents and discovery of new bioactive compounds.