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The transfer of extended spectrum β-lactamase (ESBL)-genes occurs frequently between different bacteria species. The aim of this study was to investigate the impact of nutrition related stress factors on this transfer. Thus, an Escherichia coli donor and a Salmonella Typhimurium recipient were co-incubated for 4 h in media containing different levels of the stress factors’ pH, osmolality, copper, zinc and acetic, propionic, lactic, and n-butyric acid, as well as subtherapeutic levels of cefotaxime, sulfamethoxazole/trimethoprim, and nitrofurantoin. Conjugation frequencies were calculated as transconjugants per donor, recipient, and total bacterial count. A correction factor for the stress impact on bacterial growth was used. Acetic, lactic, and n-butyric, acid, as well as pH, showed no significant impact. In contrast, increasing concentrations of propionate, zinc, copper, and nitrofurantoin, as well as increased osmolality reduced conjugation frequencies. Sulfamethoxazole/trimethoprim and cefotaxime showed increased transconjugants per donor, which decreased after correction for stress. This study showed, for the model mating pair, that conjugation frequencies decreased under different physiological stress conditions, and, thus, the hypothesis that stress factors may enhance conjugation should be viewed with caution. Furthermore, for studies on in vitro gene transfer, it is vital to consider the impact of studied stressors on bacterial growth.