A new antiviral antibiotic SF-2140 was found in the culture broth of Actinomadura sp. SF-2140 isolated from a soil sample in Hyogo Prefecture, Japan. This study reports its isolation, properties, structural elucidation, antiviral activity, antibacterial activity, and toxicity. The antibiotic was isolated via column chromatography (Diaion HP-20, silica gel, Sephadex LH-20) and crystallization. Structural elucidation using spectral data (MS, NMR, IR, UV), acid hydrolysis, and derivative preparation revealed it to be methyl (3-cyanomethyl-4-methoxyindol-1-yl 4-deoxy-α-D-lyxo-hexopyranosid)uronate. In vitro antiviral tests showed proliferation-inhibiting and inactivation activities against several influenza virus strains (e.g., A/PR-8/34, A/FM-1, Horse/Miami) with high inactivation indices. In vivo, oral administration to mice infected with influenza virus A/PR-8/34 resulted in a higher survival rate than amantadine. SF-2140 exhibited weak antibacterial activity against Gram-positive and negative bacteria. No acute toxicity was observed in mice at 2000 mg/kg intraperitoneally.