The principal targets for antibacterial agents reside at the cytoplasm and cytoplasmic membrane, damage to other structures often arising from initial events at these loci. The gram-negative bacteria offer a complex barrier system to biocides and antibiotics, regulating, and sometimes preventing, their passage to target regions. Routes of entry differ between hydrophobic and hydrophilic agents, often with a structure dependency; specialized uptake mechanisms are exploited and portage transport can occur for pro-drug antibacterials. Uptake isotherms offer insight into the sorption process and can sometimes shed light on biocide mechanisms of action. The multi-component barrier system of gram-negative bacteria offers opportunities for phenotypic resistance development where partitioning or exclusion minimizes the delivery of an antibacterial agent to the target site. Active efflux processes are recognized as increasingly relevant mechanisms for resistance, potentially offering routes to biocide:antibiotic cross-resistance. These mechanisms may be targeted directly in an attempt to compromise their role in microbial survival.