A series of gold(i) complexes with two N-heterocyclic carbene ligands (biscarbene gold complexes) were prepared and evaluated for their effects against cancer cells and pathogenic bacteria. Proliferation inhibition was observed in cancer cells and in Gram-positive bacteria, whereas Gram-negative bacteria were less sensitive towards the compounds. The protein binding and cellular uptake were quantified and the combined results indicated a strong correlation between cellular bioavailability and antiproliferative effects. The biscarbene gold complexes inhibited bacterial and mammalian TrxRs with low to moderate potency. However, based on the obtained structure-activity-relationships and the high cellular accumulation levels, TrxR inhibition can be considered as a relevant contributor to the cellular pharmacology of biscarbene gold(i) complexes.