Propolis compositions are highly variable, depending on the geographic region and the season of collection. In this study, propolis samples from seven different regions of Cyprus were studied for the first time by means of chemical content and biological activities. Secondary metabolite composition was determined by LC-HRMS. While the major flavonoids found were isosakuranetin, naringenin, rhamnocitrin, diosmetin, chrysin and acacetin, interestingly verbascoside, a phenylethanoid glycoside, and chlorogenic acid were identified as the major compounds in the ethanol-water extracts. alpha-Pinene was detected as the major compound of propolis extracts according to the volatile compositions via GC-MS. Karaoglanoglu and Tirmen extracts, presenting different chemical profiles, exerted enormous cytotoxic activity by MTT assay (IC50: 2.36-11.56 mu g/mL; 1.44-9.33 mu g/mL, respectively). The highest iNOS inhibition potential was detected in the Karpaz extract (IC50:2.6 mu g/mL) in LPS induced RAW 264.7 cells whereas the Guzelyurt sample demonstrated remarkable antioxidant (88.82 +/- 0.10%) and antimicrobial activities (with a MIC value of 31.2 mu g/mL against S. aureus, S. epidermidis, E. faecium, and E. faecalis).