Garlic (Allium sativum L.) is among the most important vegetable crops used in diet because of its sensory properties and positive health effects. Local producers in Istria (Croatia) have preserved old garlic ecotypes primarily for domestic consumption. Such ecotypes are adapted to specific agro-ecological conditions of Istria and their biochemical properties have not been investigated yet. The aim of this study is to determine the diversity of Istrian garlic ecotypes based on their biochemical profile for further selection of ecotypes with higher biological value in breeding programs. Garlic bulbs were collected from nine locations in Istria and analyzed after two months by chromatographic and spectrometric techniques. Volatiles diallyl sulfide, methyl allyl disulfide, diallyl disulfide, methyl allyl trisulfide, and diallyltrisulfide showed less variability among ecotypes, while total phenolics ranged from 28.5 to 69.5 mg gallic acid equivalents/100 g fresh weight. Two ecotypes and a cultivar abundant in phenolics also showed high antioxidant capacity measured by FRAP or DPPH assays. Several ecotypes stood out with higher dry matter content indicating their potential for longer storage. It was assumed that the determined variability in composition could be partly attributed to different genotypes and growing conditions. This research can contribute to the preservation of garlic biodiversity in Istria as well as to the establishment of a garlic breeding program.