Biofilm production by 221 Staphylococcus aureus isolates from 45 dairy herds was evaluated. Isolates were from composite milk of 117 cows, from teat skin of 70 cows, and from 34 milking machine unit liners. Of S. aureus from milk samples, 41.4% were biofilm producers, as compared to 24.7 and 14.7% of the isolates collected from skin and liners. Pulsed field gel electrophoresis (PFGE) best categorized S. aureus biofilm producers as compared to phage typing and binary typing. PFGE types that were significantly associated with isolation from milk as opposed to teat skin or liners, had isolates that were more likely to produce biofilm than PFGE types that were isolated from milk, skin and liners at similar frequencies. By contrast, PFGE type A was significantly associated with isolation from teat skin and had few biofilm producers. PFGE type Q, which is exclusively a milk, isolate produced more biofilm as evidenced by absorbance values. Given S. aureus that are associated with milk are more likely to produce biofilm as compared to extramammary sources (teat skin and milking unit liners), suggests that biofilm production is a risk factor for infection. © 2005 Elsevier B.V. All rights reserved.