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One hundred and six fungal strains were isolated from different environmental samples (fresh olive oil cake, exhausted olive oil cake, black olive, rancid butter samples, rotten bread and Roquefort) collected from the region of Meknes, Morocco (coordinates: 33°53′42″N 5°33′17″W). Yeast isolates were tested for their esterase production ability using a qualitative method based on Tween agar plate assay. Enzymatic activity was also confirmed by a quantitative method relying on esterase production in liquid medium (6 days at 28°C with shaking). Molecular characterization of the selected esterase-producing yeasts was performed by sequencing the internal transcribed spacer 1 (ITS1), 5.8S and ITS2 region of the rDNA. A total of five different species were identified in this study: Candida aaseri (LE.26, LE.27 and LE.31 strains), Wickerhamomyces anomalus (LE.106, LE.112 and LE.115 strains), Metschnikowia rancensis (LE.153 strain), Pichia sp., (LE.102) and Rhodotorula mucilaginosa (LE.171 strain). Esterase production in C. aaseri and W. anomalus was found to be straindependent, while for M. rancensis this represents the first study reporting this species as an esterase producer.