Here, we present a detailed characterisation of rhodamine B-containing magnetoliposomes (FLU-ML), emphasising the dependence of their fluorescence properties on the presence of iron oxide cores, and the molar fraction of the fluorophore. The magnetoliposome types used exist as colloidally stable, negatively charged clusters with an average hydrodynamic diameter of 95 nm. The molar rhodamine B fractions were 0.67 % and 1.97 %. Rhodamine B normalised fluorescence, quantum yields and fluorescence lifetimes were substantially reduced by inner filter effects as the magnetoliposome concentration is increased, by increasing molar rhodamine B fraction, and by quenching originating from the iron oxide cores. MR relaxometry at 3 T revealed extremely high r2 relaxivities (440 to 554 s-1mM-1) and moderately high r1 values (2.06 to 3.59 s-1mM-1). Upon incubating human prostate carcinoma (PC-3) cells with FLU-ML, a dose-dependent particle internalisation was found by MR relaxometry. In addition, the internalised FLU-ML were clearly visible by fluorescence microscopy. At the FLU-ML concentrations used (up to 3 × 103 M Fe) cell viability was not substantially impaired. These results provide valuable insights on the fluorescence properties of bimodal magnetoliposomes and open promising perspectives for the use of these materials as a platform technology for advanced functional and molecular MR and optical imaging applications. Copyright © 2012 John Wiley & Sons, Ltd.