Increased bacterial resistance to traditional antimicrobial agents has prompted the use of natural products with antimicrobial properties such as propolis, extensively employed since ancient times. However, the chemical composition of propolis extracts is extremely complex and has been shown to vary depending on the region and season of collection, due to variations in the flora from which the pharmacological substances are obtained, being therefore essential for their antimicrobial activity to be checked before use. For this purpose, we evaluate the in vitro antimicrobial and anti-biofilm activity of a new and promising Spanish ethanolic extract of propolis (SEEP) on Streptococcus mutans and Streptococcus sanguinis, responsible, as dominant 'pioneer' species, for dental plaque. Results reveal that S. sanguinis is more sensitive to SEEP, slowing and retarding its growth considerably with lower concentrations than those needed to produce the same effect in S. mutans. SEEP presents concentration- and time-dependent killing activity and, furthermore, some of the subinhibitory concentrations employed increased biofilm formation even when bacterial growth decreased. Mono and dual-species biofilms were also inhibited by SEEP. Findings obtained clearly show the relevance of using biofilm and subinhibitory concentration models to determine optimal treatment concentrations.