Bacterial conjugation is one of the key mechanisms through which antibiotic resistance genes are disseminated. However, few studies have investigated the relative contributions or interplay between the plasmid, donor and recipient cells on conjugation frequencies, with the majority focusing only on each in isolation. Here, we aimed to investigate the effects of these relationships on conjugation frequencies across 6 clinically relevant plasmid Inc types in both laboratory and clinical ST131 Escherichia coli strains. Our results support previous data on the importance of plasmid-borne factors in determining conjugation frequency but also indicate that the relationship between all three factors plays a vital role in conjugation, with conjugation frequency differences of over 50000-fold observed by varying just one factor. Significant differences in conjugation frequencies were also observed between the different directions of transfer between the same two strains, highlighting the importance of both the donor and recipient. We also present a novel infant mouse model for measuring conjugation frequency in vivo and demonstrate that the influences of the donor and recipient strains in vitro also translate to in vivo differences. These results should be taken into account when considering plasmid epidemiology, and also point to potential new targets to inhibit the spread of resistance.