Biofilm formation on stainless steel by Salmonella enterica serovar Enteritidis PT4 during growth in three different nutritious conditions was studied. The ability of micro-organisms to generate biofilms on the stainless steel surfaces was studied for a total period of 18 days at 20 °C, under three different experimental treatments: (i) growth medium (tryptone soy broth) was not refreshed (no further nutrients were provided) during the incubation period, (ii) growth medium was renewed every 2 days and (iii) growth medium was renewed every 2 days and at the same time the planktonic cells from the old medium were transferred to the new fresh medium. It was found that biofilms developed better and a higher number of adherent cells (ca. 10⁷ cfu/cm²) were recovered when the organism was grown in periodically renewed nutrient medium than when the growth medium was not refreshed. Regardless of the availability of nutrients, biofilm development was better (range 2–3 logs greater) when coupons were not totally covered by the growth medium and part of the surface was exposed to the air–liquid interface, than when coupons were submerged in the medium. The results suggest that existence of air–liquid interface and adequate nutrient conditions provide the best environment for Salmonella Enteritidis PT4 biofilm formation on stainless steel. The possible role of stationary phase planktonic cells in biofilm development by sessile/attached microbial cells is also discussed.