Harvesting plays a major role in the virgin olive oil production line, being the most expensive single component, but also due to its significant effect on the whole year's produce. Previous studies have focused on the effects of harvest timing on either oil yield or quality. Here we determined the separate and combined effects of harvesting date, fruit maturation, cultivar and fruit load on olive oil quality and quantity. Cultivars typical to the Middle East region were selected: the traditional cv. Souri and the newer cv. Barnea, grown under intensive conditions. The results demonstrate fundamental differences between the two cultivars with respect to harvest strategy. In high-yielding ‘Barnea’, oil accumulation continued throughout the ripening season resulting in increasing yield of oil with time while maintaining high quality. Hence, exploiting the production potential in ‘Barnea’ requires late harvest and advanced fruit maturity. However, in heavily loaded ‘Souri’, oil accumulation was accompanied by early massive shedding of fruits. Furthermore, late harvest and advanced maturation in ‘Souri’ were associated with a sharp increase in free fatty acids combined with a rapid decline in polyphenol content, and in MUFA to PUFA and saturated to unsaturated fatty acid ratios, all resulting in loss of oil quality. Rapid decline in oil yield coupled with deterioration of oil quality call for early harvesting at low maturity index in ‘Souri’. In medium-yielding trees of both cultivars, maturation progressed more rapidly, resulting in earlier harvest to utilize optimal oil potential.