The aim of the study was to examine heavy metal tolerance (Cd2+, Zn2+, Ni2+ and Cu2+) of single- and mixed-species biofilms (Rhodotorula mucilaginosa and Escherichia coli) and to determine metal removal efficiency (Cd2+, Zn2+, Ni2+, Cu2+, Pb2+ and Hg2+). Metal tolerance was quantified by crystal violet assay and results were confirmed by fluorescence microscopy. Metal removal efficiency was determined by batch biosorption assay. The tolerance of the mixed-species biofilm was higher than the single-species biofilms. Single- and mixed-species biofilms showed the highest sensitivity in the presence of Cu2+ (E. coli-MIC 4 mg/ml, R. mucilaginosa-MIC 8 mg/ml, R. mucilaginosa/E. coli-MIC 64 mg/ml), while the highest tolerance was observed in the presence of Zn2+ (E. coli-MIC 80 mg/ml, R. mucilaginosa-MIC 161 mg/ml, R. mucilaginosa-E. coli-MIC 322 mg/ml). The mixed-species biofilm exhibited better efficiency in removal of all tested metals than single-species biofilms. The highest efficiency in Cd2+ removal was shown by the E. coli biofilm (94.85%) and R. mucilaginosa biofilm (97.85%), individually. The highest efficiency in Cu2+ (99.88%), Zn2+ (99.26%) and Pb2+ (99.52%) removal was shown by the mixed-species biofilm. Metal removal efficiency was in the range of 81.56%–97.85% for the single- and 94.99%–99.88% for the mixed-species biofilm.