Seven yeast strains were isolated from Tunisian dates. The strains were identified by sequence analysis of the D1/D2 domain of the nuclear large subunit (LSU) rRNA gene. Based on this all strains in the study were almost identical with that of the type strain of Wickerhamomyces subpelliculosus (CBS 5767) indicating that they belong to this species. All strains were characterized physiologically and biochemically. All strains grew in the presence of 50 % sucrose, 10 % sodium chloride and at 42 °C. The potential of these yeasts as biocontrol agent against mycotoxigenic Penicillium species inhabiting date, was evaluated. All yeast strains inhibited the growth of P. citrinum P10 and P. chrysogenum C17 previously isolated from dates, with inhibition percentages ranging between 43.6 % and 70.3 % on dual culture plate assays. Moreover, the volatile compounds (VCs) produced by these yeasts inhibited the mycelial growth rate and sporulation of both fungus strains, up to 76.5 and 100 %, respectively, on inverted culture plate assay. The VCs of W. subpelliculosus strains Y4 and Y24, which exhibit strong inhibitory activity against toxigenic Penicillium, were determined by head-space solid-phase microextraction (HS-SPME) combined with gas chromatography coupled with mass spectrometry (GC–MS) analysis. Results revealed significant levels of alcohols (27.36 % for Y4 and 23.35 % for Y24) and esters (66.19 % for Y4 and 75.82 % for Y24). Their significant bioactivity, along with the lack of reported adverse effects on consumer health or the environment, makes them a sustainable and effective alternative to synthetic fungicides for the biocontrol of mycotoxigenic Penicillium affecting stored dates.