Endophytic yeasts were isolated from different organs of 18 plant species collected in Uzbekistan. More than 50 yeast isolates were obtained, of which 41 were identified using Matrix-Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF MS), while three strains were further confirmed by molecular methods based on 18S rRNA and ITS region sequencing. The identified genera included Pichia, Meyerozyma, Metschnikowia, Hanseniaspora, Kluyveromyces, and Naganishia. The antimicrobial activity of ethyl acetate extracts obtained from lyophilized whole cultures of 29 selected strains was evaluated against Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis), Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli), and the pathogenic yeast Candida albicans using the agar well diffusion method. Several isolates exhibited pronounced and broad-spectrum antimicrobial activity, with Metschnikowia pulcherrima 02, Meyerozyma guilliermondii 03, Pichia kudriavzevii 66, and isolate 023 showing the highest inhibition zones. To elucidate the biochemical basis of the observed antimicrobial effects, the most active strains were subjected to reverse-phase Liquid Chromatography-Tandem Mass Spectrometry (Nano-LC-MS/MS) analysis. Bioactive metabolites with known antimicrobial properties, including alternariol, andrastin D, altersolanol A, fumiquinazoline F, 1,4-naphthoquinone and lanosterol were putatively identified. The presence of these metabolites suggests that the antimicrobial activity of the investigated endophytic yeasts may be associated with metabolite production. This study represents one of the first reports on the isolation and metabolite profiling of endophytic yeasts from plants in Uzbekistan and the detection of these metabolites suggests that the observed antimicrobial activity may be associated with bioactive secondary metabolites produced by the isolates.