Antibiotic resistance is a global health crisis accelerated by conjugative plasmids. Incompatibility P plasmids (IncP-1) are major contributors to antibiotic resistance because they are conjugative, broad host range and are stably maintained in diverse organisms. RP1 (IncP-1 plasmid) and R91 (IncP-10 plasmid) were identified in two Pseudomonas aeruginosa strains in a Birmingham hospital. R91 reduces the conjugal transfer of RP1, a phenomenon known as fertility inhibition. Understanding the mechanism of fertility inhibition could help to reduce conjugation to combat antibiotic resistance. We sequenced and characterised R91-5 (a derepressed mutant of R91) and used transposon mutagenesis to identify the three genes (fipB1, fipB2 and fipB3) responsible for fertility inhibition of RP1. We show through genetic manipulation and studying protein interactions that the RP1 coupling protein, TraG, is targeted by the fertility inhibition genes. This improved understanding of the fertility inhibition of IncP-1 plasmids and will help to us slow the spread of antibiotic resistance.