Bacterial resistance to the antiseptic chlorhexidine (CHX), is a growing problem, recently shown to be caused by deleterious mutations to the phospholipid transport system component (mlaA) as well as efflux pump overexpression. Comparisons of CHX resistance mechanisms, such as porin deletions (ompCF), and over-expressed efflux pumps (acrB, qacE, aceI), are lacking and may be distinguishable using antiseptic rapid fluorescent dye testing assays. Using E. coli K-12 CHX adapted isolates (CHXR1), gene deletion mutants, and over-expressed transformants the phenotypes of these CHX resistance genes were compared using antimicrobial susceptibility tests (AST), rapid fluorescent propidium iodide dye-based membrane integrity assays (RFDMIA), and scanning electron microscopy (SEM). AST findings showed CHXR1, ΔacrB, ΔompCF, and transformants pCA24N-aceI and pCA24N-mlaA conferred greater (two to fourfold) MIC changes when compared to matched controls. Examination of these mutants/transformants using CHX RFDMIA showed that porin dual-deletions (ΔompCF) and mlaA alterations (ΔmlaA; pCA24N-mlaA, CHXR1) were distinguishable from controls. Results for over-expressed (pMS119EH-aceI) and deleted (ΔacrB) efflux pump RFDMIA could not be distinguished with propidium iodide, only with ethidium bromide, suggesting propidium iodide is better suited for detecting porin and mlaA associated CHX resistance mechanisms. SEM of CHXR1 and unadapted E. coli cells exposed to increasing CHX concentrations revealed that CHX does not visibly damage cell envelope integrity at any tested concentration but did identify elongated CHXR1 cells. ΔmlaA confers similar levels of CHX resistance as efflux overexpression and porin deletions, however, only outer membrane-altering porin and mlaA deletions can be reliably distinguished using RFDMIA.