Background <p>Hemolymph, as mediator of immune responses and nutrient circulation, can be used as physiological marker of an insect’s health, environmental quality or ecological adaptations. Recent studies reported intraspecific variation in protein contents and biological activities of the hemolymph of honey bees related to their diet. Here we measured interspecific variation in three common bee species in the Mediterranean Basin with contrasting ecologies: <i>Apis mellifera</i>, <i>Megachile sicula,</i> and <i>Xylocopa pubescens</i>.</p> Results <p>Despite all the bees were collected in the same area, we found important quantitative and qualitative variations of hemolymph extracts across species. Samples of <i>A. mellifera</i> and <i>M. sicula</i> had much higher protein concentration, anticancer, antimicrobial and antioxidant activities than samples of <i>X. pubescens.</i></p> Conclusions <p>This descriptive study suggests life history traits of bee species have strong influences on their hemolymph properties and call for future large scale comparative analyses across more species and geographical areas.</p>