Different filamentous fungi isolated from molasses and jams (kiwi and fig) were screened for fruc-tooligosaccharides (FOS) producing activity. Two strains, identified as Penicilium sizovae (CK1) and Cladosporium cladosporioides (CF(2)15), were selected on the basis of the FOS yield and kestose/nystose ratio. In both strains the activity was mostly mycelium-bound. Starting from 600 g/L of sucrose, maximum FOS yield was 184 and 339 g/L for P. sizovae and C. cladosporioides, respectively. Interestingly, the highest FOS concentration with C cladosporioides was reached at 93% sucrose conversion, which indicated a notable transglycosylation to hydrolysis ratio. The main FOS in the reaction mixtures were identified by HPAEC-PAD chromatography. C cladosporioides synthesized mainly 1-kestose (158 g/L), nystose (97 g/L), F-1-fructosylnystose (19 g/L), 6-kestose (12 g/L), neokestose (10 g/L) and a disaccharide (34 g/L) that after its purification and NIVIR analysis was identified as blastose [Fru-beta(2 -> 6)-Glc]. P. sizovae was very selective for the formation of F-1-FOS (in particular 1-kestose) with minor contribution of neoFOS and negligible of levan-type FOS.