Fresh tofu is a traditional Asian soybean-derived food product which has become increasingly popular worldwide, however, little is known on its microbial ecology and the microbial groups that can cause its deterioration. In this study, the microbiological characteristics of ready-to-eat fresh tofu produced by a small company in Northern Italy were investigated to unveil the abundance and composition of the microbiota and the identity and provenance of the specific spoiling microorganisms (SSO). Culture-dependent (RAPD-PCR and 16S rRNA gene sequencing) and culture-independent (PCR-DGGE) analyses were carried out on soybean lots and derived fresh tofu samples collected in different months and on two samples subjected to thermal abuse to simulate the interruption of the cold chain. Lactic acid bacteria species predominated in correctly preserved fresh tofu and bacterial species able to grow after thermal abuse mostly derived from the raw materials and belonged to heat labile species, thus suggesting that a more efficient heat treatment can increase the shelf-life of the product. Possible safety concerns could come from Streptococcus lutetientis, isolated from the artificially altered tofu, Clostridium spp. highlighted by culture-independent approach, and from Weissella confusa, but the majority of dominant bacteria identified belonged to non-pathogenic species