Rape (Brassica sp.) unifloral honey from Croatia was characterized by certain physicochemical parameters, micro- and macroelement content, and pollen spectrum, as determined in 21 honey samples. The Brassica sp. pollen type was predominant in the analyzed samples and ranged between 60 and 98%, with Trifolium spp., Robinia pseudoacacia, Rosaceae, Helianthus annuus, Salix spp., and Taraxacum officinale as the main accompanying pollen types. The electrical conductivity mean value was 0.22 ± 0.05 mS/cm and the glucose/fructose ratio mean value was 1.1 ± 0.07, whereas sucrose was absent in the samples. The most abundant macroelement was potassium (K) (268.49 mg/kg), followed by phosphorus (P) (60.23 mg/kg), calcium (Ca) (54.02 mg/kg), sodium (Na) (22.52 mg/kg), sulfur (S) (15.79 mg/kg), and magnesium (Mg) (12.58 mg/kg). Toxic elements were mainly bellow the LODs; only arsenic (As) concentration was detectable in higher amount (0.233 mg/kg), which may be related to the high arsenic concentration in the soil and groundwater of eastern Croatia. The differences between the two harvesting seasons observed in a large number of elements could be related to climatic and soil conditions and different nectar yields originating from the associated plant species.