Aims: The antibiofilm activity of endophytic fungus Nigrospora sphaerica CL-OP30 isolated from Swietenia macrophylla King was investigated. Methodology and results: The ability of the fungal endophytic crude extract to impede Streptococcus mutans biofilm formation was preliminarily screened with Congo red agar test. It was proven that S. mutans biofilm formation was hindered on the agar supplemented with the fungal endophytic crude extract. The antibiofilm activity of the fungal endophytic crude extract was evaluated using a microtiter plate method on both initially formed and preformed biofilm. Antibiofilm activity was recorded in a concentration-dependent pattern whereby higher concentrations reduced biofilm formation better than the lower concentrations of extract for both initially formed and preformed biofilm. The architecture of biofilm tested with fungal endophytic crude extract was also observed. Visualization under a light microscope and SEM revealed that the adherence of S. mutans biofilm treated with fungal endophytic crude extract was significantly reduced in both initially formed and preformed biofilm. In addition, observation under SEM showed that the matrices surrounding the bacterial cells were disintegrated and bacterial cells in biofilm completely lost their original shape. The overall data demonstrated that the ethyl acetate N. spaherica CL-OP30 crude extract showed good antibiofilm activity. Conclusion, significance and impact of study The antibiofilm study suggested the potential of N. sphaerica CL-OP30 crude extract against S. mutans biofilm by disrupting the biofilm formation, the disintegration of matrices surrounding the biofilm and responsible for the formation of irregular cell shape. This extract may have a promising potential to be developed as an antibiofilm agent.