Urban green infrastructure (UGI) plays a fundamental role in the provision of ecosystem services (ES), especially in highly urbanised environments. Among these, regulating ecosystem services (RES) are often neglected in ES assessments due to the difficulties associated with their biophysical quantification, despite their critical role. Methodological choices in ES assessment can significantly affect their estimation. This study compares two widely used tools from the i-Tree suite, i-Tree Eco and i-Tree Canopy, both designed to quantify RES provided by UGI. A field-based intensive sampling campaign was conducted in an urban park in the municipality of Toruń (Poland) to create the complete inventory required by i-Tree Eco. This tool relies on detailed tree measurements and provides comprehensive estimates of RES, including key ES such as carbon sequestration and storage, air pollutant removal, avoided runoff, and many others, resulting in very detailed outputs. In contrast, i-Tree Canopy estimates canopy cover and derives RES through manual interpretation of randomly distributed points over high-resolution aerial imagery. It estimates the same major RES of i-Tree Eco through simplified inputs. Requiring no field data, it allows rapid assessments suitable for large-scale applications and temporal comparisons. When applied to the same study area, the two tools showed consistent trends and patterns in RES provisioning, although systematic differences emerged in the absolute magnitude of the results: air filtration in i-Tree Eco was 336.03 kg vs 143.49 kg in i-Tree Canopy, carbon storage 211.2 t vs 174.4 t, and runoff 38.3 kl vs 168.7 kl. In conclusion, the findings indicate that i-Tree Eco and i-Tree Canopy should be regarded as complementary rather than alternative approaches, each being better suited to specific assessment objectives and spatial or temporal scales. These results emphasise the importance of methodological choices in urban ES assessments and provide useful insights for supporting more informed UGI management.