The current study aims to enhance glycerol production using UV-mutagenesis of the marine yeast Wickerhamomyces anomalusHH16 isolated from marine sediment collected from South Sinai Governorate, Egypt. Besides optimization of the culture conditions and analyzing the kinetic parameters of growth and glycerol biosynthesis by the mutant strain were studied. The marine yeast isolate HH16 was selected as the front runner glycerol-producer among all tested isolates, with glycerol yield recorded as 66.55 gl−1. The isolate was identified based on the phenotypic and genotypic characteristics of W. anomalus. The genotypic characterization based on the internal transcribed spacer (ITS) sequence was deposited in the GenBank database with the accession number MK182824. UV-mutagenesis of W. anomalusHH16 by its exposure to UV radiation (254 nm, 200 mW cm−2) for 5 min; increased its capability in the glycerol production rate with 16.97% (80.15 g l−1). Based on the kinetic and Monod equations, the maximum specific growth rate (μmax) and maximum specific glycerol production rate (vmax) by the mutant strain W. anomalusHH16MU5 were 0.21 h−1and 0.103 g g−1, respectively. Optimization of the fermentation parameters such as nitrogen source, salinity and pH has been achieved. The maximum glycerol production 86.55 g l−1has been attained in a fermentation medium composed of 200 g l−1glucose, 1 g l−1peptone, 3 g l−1yeast extract, and 58.44 g l−1NaCl, this medium was adjusted at pH 8 and incubated for 3 days at 30° C. Moreover, results indicated the ability of this yeast to produce glycerol (73.33 g l−1) using a seawater based medium. These findings suggest the applicability of using the yeast isolate W. anomalusHH16MU5 as a potential producer of glycerol for industrial purposes.