The natural antibiotic susceptibility of 139 Escherichia coli strains (including 18 enterohemorrhagic E. coli), 73 Shigella strains (S. sonnei (n = 37), S. flexneri (n = 29), S. boydii (n = 6), S. dysenteriae (n = 1)), 23 E. vulneris, and 20 E. hermannii strains toward 71 antibiotics was examined. MICs were determined using a microdilution procedure. All examined taxa were naturally sensitive/intermediate toward tetracyclines, aminoglycosides, some penicillins (amoxycillin/clavulanate, ampicillin/sulbactam, piperacillin [with and without tazobactam], mezlocillin, azlocillin), cephalosporins, carbapenems, monobactams, quinolones, trimethoprim, cotrimoxazole, and chloramphenicol and were naturally resistant/intermediate toward benzylpenicillin, oxacillin, macrolides, lincosamides, glycopeptides, rifampicin, and fusidic acid. No differences in natural antibiotic susceptibility were seen between enterohemorrhagic and other E. coli strains. Likewise, with one exception, no significant differences in natural antibiotic susceptibility were seen either among the Shigella subgroups or between Shigella and E. coli. The natural population of S. flexneri was slightly more susceptible to chloramphenicol than the natural populations of other taxa within the Shigella-E. coli complex. E. vulneris and E. hermannii showed susceptibility patterns to many antibiotics similar to Shigella and E. coli, but there were other antibiotics toward which there were significant differences in natural susceptibility. E. vulneris and E. hermannii were less susceptible to nitrofurantoin and slightly more susceptible to several aminoglycosides than E. coli and Shigella. E. hermannii was the only species that was naturally resistant/intermediate to ticarcillin and amoxycillin (DIN standard). The addition of clavulanic acid to the latter resulted in a decrease of seven twofold dilution steps (E. vulneris: four twofold dilution steps, E. coli/Shigella: two twofold dilution steps) of the MICs of the natural population. With the exception of cefoperazone and cefepime, E. hermannii was more susceptible to cephalosporins than strains of the other species. E. vulneris was the species most susceptible to ticarcillin and the only species that was highly resistant to fosfomycin (MIC > 256 micrograms/mL). The antibiotic susceptibility to fosfomycin was also unique for E. hermannii (MIC 32-128 micrograms/mL), whereas the natural populations of E. coli and Shigella showed lower MIC values. The data of this study represent an assessment of the natural susceptibility of strains of Escherichia spp. and Shigella subgroups to a wide range of antibiotics. These databases can be used for the validation of antibiotic susceptibility test results of Escherichia spp. and shigellae. The conformity of the natural antibiotic susceptibility test results not only among the Shigella subgroups but also between Shigella and E. coli support the status of Shigella as a subgroup of the species E. coli.