Previously, antibiotic AT-265 (proposed as 5'-O-sulfamoyl-2-chloroadenosine) lacked direct configuration proof. We isolated two nucleoside antibiotics from a Streptomyces sp. (soil sample collected in Hamamatsu-shi, Shizuoka-ken, Japan): one identical to AT-265 (designated dealanylascamycin, 2) and the other its alanyl derivative (ascamycin, 1). Their structures were determined using spectral data (FAB mass spectrometry, 1H/13C NMR) and chemical conversions—including hydrolysis of ascamycin to L-alanine, dealanylation of 1 with Xanthomonas citri cells to produce 2, and conversion of 2 to 2-chloro-9-β-D-ribofuranosyladenine—which confirmed absolute configurations. Ascamycin (1) was established as 9-β-[5-O-(N-L-alanyl)sulfamoyl-D-ribofuranosyl]-2-chloroadenine, and AT-265 (2) as 2-chloro-9-β-(5-O-sulfamoyl-D-ribofuranosyl)adenine. Biologically, 2 exhibited broad antimicrobial activity and extreme mouse toxicity (LD50 0.2 mg/kg, intraperitoneal), while 1 showed strikingly selective activity: minimal inhibitory concentrations (µg/ml) were 0.4 for Xanthomonas citri, 12.5 for Xanthomonas oryzae, and 12.5 for Mycobacterium phlei, with no activity against Staphylococcus, Escherichia, or Salmonella. Ascamycin’s toxicity to mice was one-sixteenth that of 2. Xanthomonas citri activated 1 via rapid dealanylation to 2, whereas resistant bacteria did not, suggesting sensitivity stems from an enzyme that activates 1 by removing the alanyl group.