Olive leaf biomass, generated as a residue of olive cultivation, represents a readily available source of phenolic compounds with recognized nutraceutical potential. However, the composition of these bioactive compounds is influenced by both genetic and environmental factors. Therefore, this study aimed to evaluate the effects of cultivar and sampling period on the biophenolic composition and mineral nutrient content of olive leaves from six cultivars maintained under comparable environmental conditions in central Dalmatia, Croatia. Leaves were collected during the harvest and pruning periods. Phenolic compounds were determined by HPLC and mineral nutrients by ICP-MS, FAAS, and UV/Vis spectrophotometry. Significant cultivar-dependent differences were observed for most analyzed phenolic compounds and mineral nutrients. Cultivar ‘Leccino’ exhibited the highest concentrations of several phenolic compounds and total phenols during the pruning period, while distinct accumulation patterns were recorded among cultivars. Ten of the thirteen individual phenolic compounds showed a significant sampling-period effect; nine were higher during the pruning period, whereas apigenin was higher at harvest. Seven of the ten mineral nutrients were significantly affected by sampling period, all of them decreasing towards pruning. Multivariate analysis identified cultivar as the dominant source of variation in both fractions, and the phenolic and mineral profiles were congruent among cultivars within each sampling period. Higher phenolic accumulation during the pruning period differed in magnitude among cultivars for 15 of the 24 variables. These findings support the potential valorization of olive leaves as a source of bioactive compounds and highlight cultivar and sampling period as important considerations for their targeted utilization.