Nine days after knocking the Czech out of the Eastbourne International in the second round, Boulter repeated the feat on Centre Court at Wimbledon, with a 3-6, 7-6(4), 6-4 win over the 2021 finalist.
Murray featured in the late match but was met by the ruthless power of John Isner, who served his way to a 6-4 7-6 6-7 6-4 victory in over three hours.
The Italian No.8 seed, who had won back-to-back grass titles during his Wimbledon preparations, said he was "heartbroken" to have to miss out. "The dream is over for this year but I will come back stronger."
The Spaniard showcased his trademark fighting spirit to overcome a tricky test from Francisco Cerundolo, advancing 6-4, 6-3, 3-6, 6-4 after three hours and 36 minutes.