To determine the extent of β-lactamase-producing bacteria in the gingival sulcus, subgingival biofilm samples of 20 healthy individuals aged between 28 and 64 years were investigated. β-Lactamase activity was assessed by the chromogenic nitrocefin method and β-Lactamase-positive isolates were characterized using commercial diagnostic kits and partial sequencing of the 16S rRNA gene. Enzyme activity was detected in 35 % of the individuals. The most prominent β-lactamase-producing organisms belonged to the anaerobic genus Prevotella. Other bacteria evident in our samples were Fusobacterium spp., Capnocytophaga sp., Streptococcus mutans, and Campylobacter rectus. In comparison to the prevalence of β- lactamase-producing subgingival bacteria in patients with refractory periodontitis, the extent of enzyme activity in the healthy gingival sulcus was reduced by ~50 %.