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 rates, with the majority focusing only on each in isolation. Here, we aimed to investigate the effects of these relationships by measuring the conjugation rates of a wide range of clinically relevant plasmid Inc types. Conjugation of these plasmids between both laboratory and clinical ST131 Escherichia coli strains, and across mating conditions, both in vitro and in vivo, were determined. Our results support previous data on the importance of plasmid-borne factors in determining conjugation rates but also indicate that the relationship between all three factors plays a vital role in conjugation, with conjugation rate differences of over 50000-fold observed by varying just one factor. This has broad implications on plasmid epidemiology, and all three factors should be taken into account when considering the spread of a plasmid by horizontal transfer.