Propolis is a natural product with many biological activities, and it has been used in many industrial areas, such as pharmaceuticals, food and cosmetics. Thus, in this study, we aim to evaluate the chemical content, antioxidant, antiviral activities, cytotoxicity and the iNOS of propolis samples collected from 39 different regions in Turkey. Phenolic, flavonoid and triterpene profiles were determined using the LC-HRMS method. The main phenolic compounds identified were flavonoids, including diosmetin, rhamnocitrin, isosakuranetin, naringenin, chrysin, 3-O-methyl-quercetin and acacetin, the main phenolic acid was found as caffeic acid. The major triterpene compounds were also detected as oleanolic acid and hederagenin. The composition of volatile compounds in the propolis samples was examined using GC–MS. α-Pinene, β-pinene and limonene were found as the main compounds in most of the samples. Cytotoxic activities against MDA-MB-231, PC-3, A549, HeLa and a non-tumor cell line HEK293 were performed using the MTT method. The iNOS was also determined using LPS induced RAW 264.7 macrophage cells. Propolis samples exhibited significant cytotoxic and nitric oxide inhibition activities with a significant range of IC50 values. The total antioxidant/phenolic capacities in TR equivalents of propolis samples were assayed by DPPH, CUPRAC, FRAP, and Folin methods. The total phenolics content in the studied propolis extracts were found as between 0.96 and 13.53 mmol TR/g. TAC of propolis samples range with respect to the CUPRAC and FRAP methods were found from 0.71 to 8.24 and 0.21 to 2.27 mmol TR/g, respectively. Most of the propolis extracts exhibited more than 80 % DPPH scavenging activities, almost similar or higher than the positive controls. Antiviral activities of the samples were measured as virucidal activity by in ovo. All propolis samples promise for the significant inhibition of avian coronavirus. To our knowledge, this study is the first detailed study in the literature that includes the chemical content and biological activities of Turkish propolis collected from different geographical regions.