By investigating the prevalence and resistance characteristics of Gram-negative bacteria from organic and conventional kept laying hens against 31 (Campylobacter: 29) different antibiotics using the microdilution method, we determined to what extent different keeping systems influence bacterial resistance patterns. For this purpose, samples from 10 organic and 10 conventional flocks in Bavaria (Germany) were investigated four times between January 2004 and April 2005. Altogether, 799 cloacal swabs and 800 eggs (contents and shells) were examined. The bacterial investigation performed with standardized cultural methods showed prevalence for all bacteria groups in about the same order of magnitude in the two different keeping systems: Salmonella spp. 3.5% (organic [org]) versus 1.8% (conventional [con]); Campylobacter spp. 34.8%org versus 29.0%con and E. coli 64.4%org versus 69.0%con. Coliforms (Citrobacter, Enterobacter, Pantoea) were only isolated in single cases. In eggs, generally less bacteria were detected, predominantly Escherichia; Salmonella and Campylobacter were only scarcely isolated. Salmonella enterica ssp. enterica serovar Typhimurium (n = 10) were resistant to up to nine, S. of the serogroup B (n = 4) up to six antibiotics. All tested Salmonella (n = 23) proved to be resistant to spectinomycin. Escherichia coli (n = 257org and 276con) from organic layers showed significant lower resistance rates and higher rates of susceptible isolates to nine agents, namely amoxicillin/clavulanic acid, ampicillin, cefaclor, cefoxitin, cefuroxime, doxycycline, mezlocillin, neomycin and piperacillin. In contrast, only two antibiotics turned out to be more effective in conventional isolates (gentamicin and tobramycin). In the case of Campylobacter jejuni (n = 118org and 99con), statistically significantly better rates were observed for isolates from organic flocks concerning imipenem and amoxicillin/clavulanic acid, whereas fosfomycin was more potent in strains from conventional flocks. Results of this study indicate that both resistance rates and mean minimum inhibitory concentrations of bacteria isolated from organic keeping systems have lower values than those from conventional ones, particularly recognizable for E. coli. Thus, organic livestock farming with its restrictions and additional requirements contributes to further effectiveness of antibiotics.