Piper betle (sirih) is a medicinal plant that has been reported for various pharmacological activities such as antifungal, antibacterial, antioxidant, anticarcinogenic and, anti-inflammatory. The researchwas carried out to study the antifungal activity of ethanolic extract from P. betle leaves. Matured leaves of P. betle were collected and processed to a fine powder before being extracted using ethanol. The first part of the research involved evaluation of antifungal activity of the ethanolic extracts using disk diffusion method at four different concentrations (5, 25, 50, 100 mg/mL) against 3 types of fungi isolated from laboratory surfaces. The sensitivities of the fungal towards the ethanolic extracts were determined by measuring the size of inhibitory zones. Results showed that highest concentration of the extract (100 mg/mL) inhibited fungal growth for all three types of isolated fungi with 0.97, 0.83, and 0.77 cm zone of inhibition for yeast, Aspergillus sp. and Penicillium sp., respectively. Low concentrations (5, 25 and 50 mg/mL) did not inhibit the fungal growth except for Penicillium sp. that showed zone of inhibition, 0.53 cm at concentration of 50 mg/mL. The second part of the research involved evaluation of fungal count utilizing effective dose of betel leaves ethanolic extract obtained from the first part of this research which is 100 mg/mL. This study found that no fungal growth on the microscope wire that has been sprayed with betel leaves ethanolic extract on Day 1, Day 4 and Day 7. For wire that been sprayed with ethanol 70%, the average of fungal count was same on Day 1 and Day 4 (5.6 x 102 cfu/mL) but increased to 9.2 x 102 cfu/mL on Day 7. In conclusion, betel leaves extract exhibit fungicidal properties that support their use as antifungal agents from natural products which are safe, easily available with no adverse effects.