Pseudomonas syringae pv. actinidiae (Psa) is a phytopathogen responsible for bacterial canker in kiwifruit plants and can be disseminated through pollen. This study aimed to evaluate the effectiveness of antimicrobial photodynamic therapy (aPDT) in the inactivation of Psa on kiwifruit pollen using New Methylene Blue (NMB) and Methylene Blue (MB) in the presence/absence of potassium iodide (KI). Pollen germination assays were also performed to evaluate if it was affected by aPDT. Higher reduction of Psa was achieved using NMB (5.0 μM) combined with KI (100 mM) in vitro (ca. 8 log CFU mL−1 after 90 min of irradiation), while NMB alone promoted a lower reduction (3.7 log CFU mL−1). The most efficient NMB concentration with KI was used to study the photodynamic efficiency of MB (5.0 μM). MB with KI photo-inactivated Psa more efficiently than NMB, causing the same bacterial reduction (ca. 8 log CFU mL−1) in half the irradiation time (45 min). Therefore, MB was selected for the subsequent ex vivo aPDT assays in pollen. Almost all the Psa cells added artificially to the pollen (3.2 log CFU mL−1) were photo-inactivated (3.1 log CFU mL−1), whereas aPDT had a low effect on pollen natural microorganisms. When KI was added, a significant increase in aPDT effectiveness was observed (4.5 log CFU mL−1). No negative effects were observed in the pollen germination after aPDT. The results show aPDT is an effective and safe method to Psa inactivation on kiwifruit pollen, and MB use is a promising alternative in the control of Psa transmission.