Proline is an amino acid that increases plant tolerance to abiotic and biotic stresses, but the effects of its application can be influenced by many factors. The present study investigated the effects of time and the number of applications of this amino acid on the yield of root parsley in field conditions. The experimental material comprised of two parsley cultivars (Petroselinum crispum (Mill.) Nyman ex A.W. Hill), ‘Halblange’ and ‘Sonata’. The parsley plants were sprayed with proline (1000 mg L−1) at growth stages determined according to the BBCH scale: BBCH 15–16 (I: 5–6 leaf phase), BBCH 41 (II: roots start to widen, diameter > 0.5 cm), and BBCH scale 42–43 (III: roots are 20–30% of the typical diameter), including I + II, II + III, and I + II + III. The time and number of proline applications affected the weight of leaves and the total and marketable yield. The amino acid spraying increased the average number of plants during harvest in the ‘Halblange’ but decreased the number in the ‘Sonata’ in all applications. Using proline twice or three times reduced the total essential oil content and modified its composition. The most beneficial effect in terms of the composition was achieved by using proline twice at stages II + III, even in ‘Sonata’, where the values were lower in the other treatments than in the control. Spraying three times did not give better results in terms of the composition and content of the essential oil. However, the use of this amino acid did not affect the total and marketable yield of the roots or the leaf weight of the parsley compared to the control. Our study showed that the time of the proline application may be more important than the number of applications, and the results may be cultivar-dependent.