Electrospun nanofibers were prepared from Rhodomyrtus tomentosa extract and poly(vinyl alcohol). The antimicrobial effect was assessed against two Gram-negative bacterial strains (Escherichia coli, Pseudomonas aeruginosa) and two Gram-positive bacterial strains (Bacillus subtilis, Enterococcus faecalis) by paper disc diffusion method. Ethyl acetate extract of R. tomentosa was selected for fabrication of nanofibers because it shows the most active antimicrobial activity with zone of inhibition ranging from 9.33 +/- 0.21 to 13.67 +/- 0.32 mm. The presence of high abundance of myricetin and rhodomyrtone might contribute to the antibiotic activity against all tested bacterial strains. The average diameter of the R. tomentosa extract/poly(vinyl alcohol) nanofibers increased from 120.4 to 214.8 nm with increasing concentration of R. tomentosa extract from 0.5% to 2.5%. The antimicrobial activity of R. tomentosa extract/poly(vinyl alcohol) nanofibers was relatively higher at concentration of the extract (1.5% and 2.5%) against all test organisms with a clear zone of inhibition 7-12 mm. The results demonstrated that R. tomentosa extract/poly(vinyl alcohol) electrospun nanofibers are an interesting platform for delivery of bioactive compounds as wound dressing or other strategies for combating bacterial infections.