The objective of this work was to identify Gram-positive psychrotrophic microorganisms in expansion tanks, and to evaluate the production of enzymes and the profile of resistance to antibiotics. Samples were collected from 32 tanks using swabs and 53 isolates were selected. The presence of Bacillus, Enterococcus, Corynebacterium, Styaphylococcus, Lactococcus and Micrococcus was observed. The isolates were evaluated for enzyme production, and high proteolytic and lipolytic activity was verified. In the experiment, 12 antibiotics were used and all isolates showed multiple resistance to the tested drugs. It is concluded that good agricultural practices in milk production are essential to reduce the multiplication of these microorganisms, so that consumers can be adequately served.