Objective To study the effect of clove oil on bacterial biofilm of Streptococcus pneumoniae. Methods The components of clove oil were determined by GC-MS mass spectrometry. The suspension of streptococcus mutans was prepared to carry out the germicidal test, and to determine the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of clove oil. The effects of different concentrations of clove oil on the formation of biofilm were observed by laser confocal microscopy, and the average fluorescence intensity of live bacteria and dead bacteria in biofilm was recorded. Results Determination of clove oil 8 components were analyzed by GC-MS mass spectrometry method, most of the Eugenol content was 48.93%, followed by the Caryophyllene 20.78%, Methylis salicylas 14.96%. Through MIC, MBC and the experimental results showed that clove oil had antibacterial activity of Proteus, and with the increase of clove oil concentration, the inhibitory effect was better. When it reached a certain concentration, it can showed bactericidal effect that the MIC and MBC were 0.3125%, 0.6250%. The clove oil also had inhibitory effect on Streptococcus mutans biofilm with the concentration depedence. When the concentration of clove oil was 2.5000%, the average fluorescence intensity (live/dead bacteria) was 0.082 ± 0.007, the proportion of living bacteria decreased obviously, and biofilm disappeared. Conclusions The clove oil not only has inhibitory effect on Streptococcus mutants, but also can inhibit and clear the biofilm formation of Streptococcus.