Root endophytic freshwater hyphomycetes Cylindrocarpon aquaticum Nils. was evaluated for its antibiotic potential against some Gram-positive (Bacillus subtilis) and Gram-negative (Agrobacterium tumefaciens, Escherichia coli, Erwinia chrysanthemum, Xanthomonas pseudomonas) bacterial strains. Antimicrobial activity was assessed with preliminary and secondary antibiotic assays. The results were found positive with the applied fungus by producing significant zone of inhibition was observed for all tested bacterial strains. In primary antimicrobial assay maximum zone of inhibition was counted against Gram-positive bacterium Bacillus subtilis (26 mm) followed by Gram-negative bacteria Xanthomonas phaseoli (24 mm) and Agrobacterium tumefaciens (23 mm) while minimum zone of inhibition was observed against Erwinia chrysanthemi and Escherichia coli (22 mm, each). Whereas, in secondary antimicrobial assay the maximum zone of inhibition was recorded against human pathogenic bacterial strain Escherichia coli (16 mm) followed by Bacillus subtilis (15 mm) and minimum inhibition was found against Agrobacterium tumefaciens (10 mm).