OBJECTIVE To characterize the genera of mycelial fungi detected in expressed human milk received at the human milk bank of Instituto Fernandes Figueira after home collection. METHODS We studied 821 expressed human milk samples randomly obtained from flasks filled by the donors at home. The possible presence of molds, yeasts and mesophilic microorganisms was investigated. A total of 48 strains of mycelial fungi were isolated from the human milk bank samples and identified through standard laboratory techniques. RESULTS Microbiological analysis revealed the occurrence of molds and yeasts in 43 samples (5.2%), with counts reaching 10(3) CFU/ml. The following microorganisms were identified: Aspergillus niger group (6.3%), Aspergillus sp. (4.2%), Paecilomyces sp. (12.6%), Penicillium sp. (60.4%), Rhizopus sp. (2.0%), and Syncephalastrum sp. (14.5%). Four samples showed the presence of more than one mycelial fungus type. CONCLUSIONS The presence of molds and yeasts in human milk manually expressed at home suggests that the hygiene conditions of the collection site may contaminate milk. Thus, when hospitalized premature babies receive the raw product, it is very important to observe the collection, storage and transport conditions in order to avoid the presence and consequences of contaminants increase.