Background: beta-Glucosidases are components of the cellulase system, a family of enzymes that hydrolyze the beta-1,4 linkages of cellulose. These proteins have been extensively studied due to the possibility of their use in various biotechnological processes. They have different affinities for substrates (depending on their source) and their activities can be used for saccharification of different types of biomass. In this context, the properties and the synergistic capacity of beta-glucosidases from different organisms, to supplement the available commercial cellulase cocktails, need a comprehensive evaluation. Results: Two beta-glucosidases belonging to GH3 family were secreted by Penicillium citrinum UFV. Pc beta Glu1 (241 kDa) and Pc beta Glu2 (95 kDa) presented acidic and thermo-tolerant characteristics. Pc beta Glu1 showed Michaelis-Menten kinetics for all substrates tested with K-m values ranging from 0.09 +/- 0.01 (laminarin) to 1.7 +/- 0.1 mM (cellobiose, C2) and k(c), values ranging from 0.143 +/- 0.005 (laminarin) to 8.0 +/- 0.2 s(-1) (laminaribiose, Lb). Pc beta Glu2 showed substrate inhibition for 4-methylumbelliferyl D glucopyranoside (MU beta Glu), p-nitrophenyl-beta-D-glucopyranoside (pNP beta Glu), cellodextrins (C3, C4, and C5), N-octil-beta-D-glucopyranoside, and laminaribiose, with K m values ranging from 0.014 +/- 0.001 (MU beta Glu) to 0.64 +/- 0.06 mM (C2) and k(m), values ranging from 0.49 +/- 0.01 (gentiobiose) to 1.5 +/- 0.2 s(-1) (C4). Inhibition constants (K) for Pc beta Glu2 substrate inhibition ranged from 0.69 +/- 0.07 (MU beta GIu) to 10 +/- 1 mM (Lb). Glucose and cellobiose are competitive inhibitors of Pc beta GIu1 and Pc beta Glu2 when pRIPPGIu is used as a substrate. For Pc beta Glu1 inhibition, K = 1.89 +/- 0.08 mM (glucose) and K = 3.8 +/- 0.1 mM (cellobiose); for Pc beta Glu2, K= 0.83 +/- 0.05 mM (glucose) and K = 0.95 +/- 0.07 mM (cellobiose). The enzymes were tested for saccharification of different biomasses, individually or supplementing a Trichoderma reesei commercial cellulose preparation. Pc beta Glu2 was able to hydrolyze banana pseudostem and coconut fiber with the same efficiency as the T reesei cocktail, showing significant synergistic properties with T reesei enzymes in the hydrolysis of these alternative biomasses. Conclusions: The beta-glucosidases from P citrinum UFV1 present different enzymatic properties from each other and might have potential application in several biotechnological processes, such as hydrolysis of different types of biomass.