The antimicrobial efficacy of two aldehyde-based biocides (glutaraldehyde, GTA, and ortho-phthalaldehyde, OPA) and two surfactants (cetyltrimethyl ammonium bromide, CTAB, and sodium dodecyl sulphate, SDS) was tested against planktonic Pseudomonas fluorescens. The antimicrobial effects were evaluated by respiratory activity as a measure of the oxygen uptake rate, adenosine triphosphate (ATP) release, outer membrane proteins (OMP) expression and cellular colour changes. The results were compared with the bacterial characteristics without chemical treatment. Tests in the presence of bovine serum albumin (BSA), in order to mimic a disinfection process in the real situation under dirty conditions, were performed according to the European Standard EN-1276. P. fluorescens was completely inactivated with OPA (minimum bactericidal concentration, MBC = 0.5 mM) and CTAB (MBC = 5 mM) and was resistant to GTA and SDS. Only CTAB promoted cellular disruption and consequent ATP release. The antimicrobial action of the chemicals tested was significantly reduced when BSA was introduced into the bacterial cultures, increasing markedly the MBC values. Additionally, the presence of BSA acted as a disruption protective agent when CTAB was applied and stimulated the bacterial respiratory activity when lower concentrations of SDS were tested. The OMP of the bacterial cells was affected by the application of both surfactants. OMP expression remained unaltered after biocide treatment. Bacterial colour change was noticed after treatment with biocides and surfactants. In summary, P. fluorescens was extremely resistant to GTA and SDS, with antimicrobial action being quenched markedly by the reaction with BSA.