Recent years have witnessed a growing interest in calendula (Calendula officinalis L.), as an oil-bearing plant whose seeds contain unique polyunsaturated fatty acids that can be used in pharmaceutical, paint and coatings industries. The influence of harvest date on seed yield and oil biosynthesis in calendula seeds has not been widely researched. A three-year field experiment was carried out to examine the effect of harvest dates at 50%, 65% and 80% seed maturity on the yield and fatty acid composition of seven calendula cultivars. The highest seed (1096–1950 kg ha−1) and oil (181–391 kg ha−1) yield and the highest oil content (16.39–20.55%) were achieved when calendula plants were harvested with 65% of mature seeds, and yields were somewhat smaller with 80% of mature seeds. The oil from calendula seeds harvested at 80% seed maturity was characterized by the highest content of α-calendic acid (43.60%–54.39%) and the lowest content of the remaining fatty acids. The Partial Least Squares Regression analysis revealed that oil (r2 = 95.8) and seed (r2 = 98.1) yield depends on the morphological features of the examined cultivars, such as the number of flower heads, percentage of morphological seed types and climatic factors (mean temperature and total precipitation during the growing season). The results clearly indicate that seed and oil yield was highest in cultivars with the highest percentage of winged and hooked seeds and the highest number of flower heads.