Conjugation of plasmids is widespread among bacteria and contributes to the spread of antibiotic resistance. In the natural environment, microorganisms predominantly exist in the form of biofilms or other bioaggregates, where they may be exposed to contaminants such as antibiotics, at subinhibitory concentrations. Bacterial cells in older biofilms have lower growth activity due to oxygen and nutrient limitation in the deeper layers of the biofilms. In batch culture, population growth eventually ceases during the stationary phase. Thus, the steady state of biofilms may resemble stationary growth phase cultures. Our objectives were to study (i) the effect of cell growth phases and (ii) subinhibitory and minimum inhibitory concentrations (MIC) of antibiotics on transconjugant formation in both batch cultures and biofilms. Additionally, (iii) the effect of variable nutrient concentrations on MIC was investigated and (iv) an optimization of RT-PCR method for the detection of traA gene (which encodes pilus biosynthesis) expression was carried out. To study the effect of cell growth phases on transconjugant formation, plate matings were carried out utilizing planktonic cultures grown to exponential or stationary phase of donor and recipient strains. The results showed that transconjugant abundance was the highest (20± 0.08%) when both plasmid donor and recipient cells were grown to the stationary phase. However, the growth phase of the donor did not seem to play a role in biofilms. When donor cells were harvested from either the exponential or stationary phase of growth, and inoculated into 24 h old recipient biofilms, there was no statistically significant difference between transconjugant abundance. A higher percentage of transconjugants was detected in plate matings when the donor was exposed to 0.5× minimum inhibitory concentration of gentamicin and additionally challenged with gentamicin at MIC. In biofilms, transconjugant formation was not enhanced when the donor cells were grown with 0.5× MIC gentamicin, and 0.5× MIC gentamicin was added to the biofilms. A decrease in nutrient concentration was associated with a decrease in the MIC. traA expression, detected using RT-PCR in plasmid donor cells grown to early exponential and late exponential phases did not coincide with an increase in transconjugants.