Overproduction of metabolites, high product yield and process economics are greatly influenced by the media composition used for growth and fermentation. The main purpose of this study is to enhance the ethanol production through statistical tool of response surface methodology (RSM) by optimizing media components for the growth and fermentation of thermotolerant isolates Kluyveromyces marxianus NIRE-K1 and NIRE-K3. Five different salts were used in the Face-centered Central Composite Design (FCCD), with the responses of biomass formation and ethanol production for growth and fermentation, respectively. Yeast extract and K2HPO4 were found to be the key media components for the growth and fermentation which is revealed from their interaction in both the yeast isolates. Further studies on batch fermentation kinetics using the optimized values of the medium composition for K. marxianus NIRE-K1 and NIRE-K3 resulted in final ethanol concentration of 17.73 (86.27% of theoretical ethanol yield) and 19.01 g l−1 (94.12% of theoretical ethanol yield), respectively. An increase in the ethanol yield and productivity by 11.36, 10.42% and 2.0, 2.7% was revealed in NIRE-K1 and NIRE-K3, respectively, as compared to our previous study.