The aims of this study were to investigate the development of bacterial resistance to eugenol, thymol, trichlorocarbanalide (TCC), didecyldimethylammonium chloride (DDDMAC) and C10-16-alkyldimethyl, N-oxides (ADMAO) and subsequent effects on antibiotic susceptibility. An agar minimum inhibitory concentration (MIC) method was used to assess the activity of the biocides against standard bacterial strains and laboratory mutants. A range of techniques including disk diffusion and gradient plate experiments were used to attempt to develop bacterial 'resistance' or tolerance to the biocides. The mutants produced were examined for cross-resistance to the other biocides and to antibiotics via disk diffusion and gradient plate MIC methods. Outer membrane proteins of the mutants were extracted and examined using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Escherichia coli triclosan-resistant mutants were not cross-resistant to eugenol, thymol, TCC, DDDMAC and ADMAO. Mutants with elevated MICs to DDDMAC (E. coli and Pseudomonas aeruginosa), thymol (E. coli) and eugenol (E. coli) were isolated, but all remained sensitive to higher concentrations of the agents. Bacteria with elevated MICs to TCC and ADMAO were not obtained. Some low-level cross-resistance between DDDMAC, eugenol and thymol was observed with the E. coli gradient plate mutants, as well as reduced susceptibility to antibiotics, most notably chloramphenicol. The lack of cross-resistance of the triclosan mutants suggested that the mode of action of triclosan is not shared with the other biocides studied. SDS-PAGE results indicated that the DDDMAC P. aeruginosa mutant had a reduced amount (or absence) of one outer membrane protein in comparison with the standard strain. In conclusion, under laboratory conditions, bacterial exposure to thymol, eugenol and DDDMAC can lead to reduced susceptibility between selected biocidal agents and antibiotics, more specifically, chloramphenicol. However, further studies are required to determine if this is of clinical significance.