Background: The Cyprus Biobank collects biosamples and medical, biological, and lifestyle information with the aim of reaching 16,500 Cypriot volunteers aged ≥18-years, by year 2026. Volunteers are both from the general population and from disease cohorts of focused research projects, who amongst others will contribute to canvas the architecture of the Cyprus human genome and study the healthy and morbid anatomy of Cypriots, a population with a unique historical and epidemiological background. The Cyprus Biobank is a research infrastructure pillar of the biobank.cy Center of Excellence in Biobanking and Biomedical Research. Methods: Within 3-years (November 2019-October 2022), 1348 participants of the general population were enrolled in the Cyprus Biobank pilot study. Extensive information was collected from each participant at enrolment, including biochemistry, complete blood count, physiological, anthropometric, socio-demographic, diet, and lifestyle characteristics. Prevalent health conditions along with medication use and family history were recorded, including 58 biomarkers based on blood and urine samples. With a systematic recruitment campaign, the Biobank is continuously increasing the number of individuals in the general population cohort and is developing separate disease cohorts of the Cypriot population. Results: The pilot study enrolled 1348 participants (579 men and 769 women), aged between 18-85 years (median 48-years). The enrollment takes 40 minutes on average, including the collection of biological samples and phenotypic information. More than half (55%) of the pilot participants are educated to college level or above. Statistically significant differences were found between men and women regarding their marital status (p = 0.01), education level (p < 0.001), and employment status (p < 0.001) but not their age (p = 0.29). The most prevalent medical conditions recorded within the studied population are hypertension (17.2%), osteoporosis (6.9%) and diabetes (6.0%). 1000 whole exomes, as the Phase-1 of the Cyprus Human Genome Project, CYPROME, has been completed. Conclusions: The Cyprus biobanking pilot study has successfully collected extensive baseline information from enrolled participants. With expanding beyond the pilot study, the Biobank will comprise a rich data resource that will be used to examine the major risk factors leading to public health burden and to develop strategies for disease prevention.