10.22376/ijpbs.2019.10.1.p1-12 Volume 11 Issue 2 2020 (April-June) The capability of bacteria such as lessThan i greaterThan Streptococcus mutans lessThan /i greaterThan to form biofilm leads to increased resistance to some conventional antimicrobial substances, indicating a need for alternative antibiotics. Therefore, the present study was carried out to investigate the anti-biofilm activity of ethyl acetate crude extract of endophytic fungus, lessThan i greaterThan Lasiodiplodia pseudotheobromae lessThan /i greaterThan IBRL OS-64, isolated from the leaf of lessThan i greaterThan Ocimum sanctum lessThan /i greaterThan against lessThan i greaterThan S.mutans lessThan /i greaterThan . The Congo red agar and semi-quantitative adherence assays were performed to determine biofilm formation and anti-biofilm activity, respectively. Meanwhile, the microscopic analysis was carried out to evaluate the efficacy of the extract in eradicating biofilm formation. The findings revealed that lessThan i greaterThan S. mutans lessThan /i greaterThan is a good biofilm producer by producing black colonies on Congo red agar, an optical density (OD) value of 0.643 on the semi-quantitative adherence assay and the fungal extract possesses anti-biofilm activity by eradicating biofilm formation on the test tube and Congo red agar. Furthermore, the extract demonstrated significant anti-biofilm activity against initial and pre-formed biofilm of lessThan i greaterThan S. mutans lessThan /i greaterThan with the highest percentage of inhibition of 89.23% and 54.14%, respectively. The light microscopy (LM) and scanning electron microscopy (SEM) photomicrographs proved that the fungal extract significantly eliminates the extracellular polysaccharide (EPS) matrices and hinder the attachment of the bacterial cells for biofilm formation. Hence, the data suggest that ethyl acetate crude extract from lessThan i greaterThan L. pseudotheobromae lessThan /i greaterThan IBRL OS-64 may be an effective anti-biofilm agent to eradicate oral cavity bacteria. Taufiq, M.M.J and Darah, I Streptococcus mutans, Lasiodiplodia pseudotheobromae, anti-biofilm activity, light microscopy and scanning electron microscopy, extracellular polysaccharide 7-18