In this study, various fruit were employed as biocontrol yeast reservoirs, and eleven distinct yeast cultures of Metschnikowia sp. belonging to 6 different species were identified with sequence-based analysis of the D1/D2 domain of 26S rDNA. For initial screening, Metschnikowia isolates were tested on Fusarium oxysporum, Botrytis cinerea, Penicillium digitatum, Penicillium expansum, and Alternaria alternata. The highest antagonism was obtained on green and blue Penicillium (83.63–100 %). All tested yeasts showed chitinase activity, while some had protease, pectinase, cellulase, β-1−3 glucanase, and gelatinase activities. Since lemons have high pectin content, three pectinase-free cultures at tested conditions with high in vitro antagonism on Penicillium were selected and used on lemons. The activities of the in vitro antifungal studies were found to be compatible with those of the in vivo, and P. digitatum's incidence was found to be higher than that of P. expansum on lemons. All tested pectinase-free Metschnikowia sp. lead to a significant reduction in the disease incidences and lesions at varying levels. The combined effect of lytic enzyme secretion, iron depletion, and volatile organic compounds (VOCs) production determined the antifungal mechanism of action. M. aff. fructicola demonstrated the highest biocontrol efficacy against Penicillium on lemons with an increasing shelf-life. The use of tentatively pectinase-negative Metschnikowia sp. as an antifungal biocontrol agent on lemons was considered for the first time. The findings will shed light on the effective use of Metschnikowia sp. as a potential biofungicide against the growth of postharvest fungal pathogens.