Propolis is a resinous substance produced by honeybees with plant ingredients, which is used to fill gaps in the hive to prevent the entry of undesirable visitors and to provide better thermal insulation. It has been used for centuries to treat different pathologies and more recently became very popular in Europe due to its antibacterial activity. Biofilm is a multi-layered cluster of bacteria embedded in an extracellular polysaccharide matrix, which is known to increase bacterial ability to colonise inert materials and to protect bacteria from body defence mechanisms and antimicrobials, contributing to the establishment of persistent infections. Bacteria of the genus Staphylococcus are responsible for a plethora of infections in humans and animals and are the main etiological agents of mastitis in ruminants. The production of biofilm by these bacteria increases their resistance to antimicrobials, greatly hindering the treatment of infections. This study aims to investigate the in vitro activity of propolis ethanol extracts (PEE) against biofilms produced by staphylococci isolated from the milk of small ruminants with mastitis. The inhibitory action on biofilm formation and the PEE ability to eliminate established biofilms were evaluated. Ten PEE were produced from seven samples of propolis harvested in several regions in Brazil (one green, two red and four brown) and three samples collected in different regions in Portugal (all brown). These PEE were assessed for biofilm formation inhibition and biofilm disruption ability on 45 biofilm producing Staphylococcus isolates (26 S. aureus, seven S. chromogenes, four S. warneri, three S. auricularis, two S. simulans, one S. caprae, one S. capitis, and one S. epidermidis) on polystyrene flat-bottom microtiter plates. All PEE showed antibiofilm activity against some Staphylococcus isolates. Generally, PEE are more effective in inhibiting biofilm formation, than in destroying the formed biofilm. According to these results, propolis deserves to be considered for the control of infections caused by biofilm producing staphylococci.