Ashton viewed with "great concern" an Israeli decision to declare an area near the Gush Etzion settlement south of Bethlehem as state land and approval of a new Jewish settlement in the southern city of Hebron, a statement from her office said.
The continued demolition of Palestinian property and the confiscation of EU humanitarian aid were also worrying, Ashton added.
Earlier this month, an EU official said Israel had demolished several EU-funded humanitarian housing shelters in a highly sensitive strip of West Bank land near Jerusalem.
"The EU calls on the Israeli authorities to reverse these decisions," Ashton said.
Such events are "not conducive to the climate of trust and cooperation needed for the current peace negotiations to succeed," she added.
Ashton also said she "condemns the recent killing of an Israeli man in the West Bank and calls for an immediate end to all acts of violence."
All sides should "show utmost restraint and responsibility in order not to jeopardize the current negotiation process," she said.
An Israeli official, who requested anonymity, slammed Ashton's comment, pinning the blame for the faltering peace talks on the Palestinians.
"It is not surprising that once more Mrs. Ashton is ignoring Palestinian responsibility in the crisis facing the discussions," the official said.
"Although the Palestinians clearly and crudely violated the agreements by unilaterally addressing the United Nations, Mrs Ashton did not see the need to react to that."
Under an agreement brokered by the United States for the resumption of the talks last July, Israel committed to releasing 104 prisoners held since before the 1993 Oslo autonomy accords in four batches.
But in March it cancelled the release of the last group of 26, triggering the ire of Palestinian president Mahmud Abbas who retaliated by seeking accession to 15 international treaties and conventions.
The crisis emerged just a month before the deadline of the talks on April 29 and as Washington was striving to extend the negotiations beyond that date.