Fructooligosaccharides (FOS) are fructose-based oligosaccharides employed as additives to improve the nutritional and technological properties of foods. The rhizosphere of inulin-accumulating plants from the Cerrado (Brazilian savanna) harbor fungi capable of synthesizing FOS from sucrose through the transfructosylating activity of β-fructosyltransferases and/or β-fructofuranosidases. Here, we investigated the ability of Penicillium janczewskii Zaleski CCIBt 3352, a fungus isolated from the rhizosphere of Chrysolaena obovata (Asteraceae), to produce FOS in a medium supplemented with sucrose concentrations of 30, 100, or 150 g L . Hydrolytic activity on sucrose was observed in culture filtrates; however, at 150 g L sucrose, the accumulation of 8 g L 1-kestose (inulin-type FOS) and 7.3 g L neokestose (neolevan-type FOS) was observed, the latter being a type of FOS not commonly produced by filamentous fungi. In addition, minor amounts of four unidentified oligosaccharides, with a high degree of polymerization, were detected. The production of FOS was also observed in enzymatic assays, indicating the presence of extracellular enzymes with transfructosylating activity in the culture filtrates. Our findings demonstrate the feasibility of isolating promising microorganisms, for the production of FOS-synthesizing enzymes, from the rhizosphere of fructan-producing plants of the Brazilian Cerrado.