A decrease among honey bee populations (Apis mellifera) in the traditional apiaries has been observed in recent years. In light of this negative phenomenon, urban beekeeping seems to be an appropriate alternative solution for the bee population in reducing the toxic effects of a large number of pesticides that are commonly used in agricultural ecosystems. Despite the rapid development of urban beekeeping, there is little information regarding the different aspects of the defense effectiveness of bees from the urban and rural areas. The study was aimed to show whether honey bees from these two locations differ in the level of the valuable biomarkers of stress exposure helpful in establishing which bees, from urban or rural areas, are under greater environmental pressure. For this purpose, foragers from an urban rooftop apiary and a traditional rural apiary were collected. The chosen biomarkers were measured in various tissues of bees. The activity of glutathioneS-transferase and acetylcholinesterase, the level of total antioxidant capacity, heat shock protein 70 (Hsp70), and defensin were selected for the analyses. In our opinion, the Hsp70 and defensin levels seemed to be important in the indication of urban multistress factors. The higher level of heat shock proteins and defensins in tissues/organs of bees from the urban apiary—in the gut (an increase, respectively, 92% and 7.3%) and fat body (an increase, respectively, 130% and 7.8%), known as targets of environmental toxins, pointed out the urban environment as highly stressful at both the individual and colony levels. In turn, high total antioxidant capacity was measured in the guts of honey bees from rural area (an increase 107%). Such a situation suggests a different mechanism of defense and specificity of rural and urban environmental stressors and also honey bees foraging activity.