Objectives Major lignocellulosic inhibitory compounds found in sugarcane-based industrial hydrolysate samples were tested in laboratory and industrial yeast strains, as well as in lactic acid bacteria, in order to verify their effects on important physiological parameters. Results Saccharomyces cereviaise SA-1, an industrial strain, stood out as compared to the remaining strains for virtually all inhibitors investigated. This strain presented the highest growth rate and the lowest lag-phase in the presence of acetic acid, levulinic acid, p-coumaric acid, ferulic acid, and HMF, when compared to the other strains. In sugarcane-based hydrolysate fermentations, both SA-1 and CEN.PK113-7D presented similar fermentation performances. Industrial isolates of contaminating lactic acid bacteria were evaluated in the presence of an inhibitory cocktail, containing a mixture of 76.6 mM acetic acid, 1.3 mM HMF, 7.1 mM furfural, and 1.9 mM p-coumaric acid. Whilst all yeast strains were unable to grow under such conditions, bacteria had an average inhibition of roughly 50% on their growth rates. Conclusions Overall, industrial strain SA-1 might be a promising microbial chassis for second generation ethanol production and for future metabolic and evolutionary engineering strategies, and for strain robustness understanding.