The effect of pre-conditioning polished and unpolished AISI 304 stainless steel surfaces with glutaraldehyde on the attachment, growth and morphology of an aerobic consortium of marine bacteria was investigated using total cell number counts, epifluorescence microscopy, Atomic Force Microscopy (AFM), Environmental Scanning Electron Microscopy (ESEM) and grazing-angle Fourier Transform Infrared (FÜR) spectroscopy. Both fully hydrated and dehydrated biofilms were studied using AFM and ESEM. Formation of the conditioning layer on steel surfaces from the culture medium, in the presence and absence of glutaraldehyde was monitored in-situ employing AFM and Grazing Angle FT1R spectroscopy. The influence of both surface area and surface energy upon the numbers of bacteria attached to polished and unpolished coupons was determined. This study has shown the influence of pretreatment of AISI 304 stainless steel with glutaraldehyde upon biofilm formation and has demonstrated the ability of AFM, ESEM and FTIR to be used as valuable tools for the in-situ investigation of the effect of biocides on bacterial biofilms.