Latest figures show the the nursing and midwifery vacancy rate stands at 4.5% - the highest ever reported.
The Royal College of Nursing (RCN) said the current situation with unfilled posts threatened patient care.
Health Secretary Shona Robison insisted staff levels within the NHS had risen to "historically high levels" under the SNP.
Figures from ISD Scotland show 2,818.9 whole-time equivalent (WTE) posts were unfilled at the end of March, a 27.5% increase from March last year.
The number of such posts lying unfilled for three months or more was up 51.3% year on year to 670.6.
During the same period, the NHS spent Â£8.4m more on nursing and midwifery bank and agency staff, paying out a total of Â£166.5m.
Vacancies for consultants have also risen year on year from 6.5% to 7.4% and there were 415.7 vacancies, of which 203.4 have lain empty for more than three months, up 38.2 from 2016.
The total number of staff working within the Scottish NHS, however, has risen 0.7% year on year to 139,430 WTE posts.
Norman Provan of the RCN warned: "If there aren't the nurses, patients won't receive the care they need."
He added: "These figures reflect the challenge faced by Scotland's NHS. Across both acute and community settings, there are simply too few nurses.
"The Scottish government can point to the increase in the number of nursing and midwifery staff, but the reality on the ground is that nurses wanting to do their very best for patients are too often coming up against the reality of vacancies in the workforce.
"Nursing morale is low and teams are struggling to recruit and retain the staff they need."
Health Secretary Ms Robison said part of the increase in vacancies was due to the creation of new posts in health boards.
She said: "Under this government, NHS staff numbers have risen to historically high levels, with more doctors and nursing staff now delivering care for the people of Scotland - including over 2,000 additional qualified nurses and midwives.
"We're committed to training and retaining our nursing staff, and earlier this year we confirmed a 4.7% increase in trainee nurses and midwives for 2017/18 - a fifth successive rise.
"We have also committed Â£450,000 over three years to reintroduce a national return to practice scheme."

Marmalade, a 13-year-old ginger cat, was found nearly four months after going missing from Tracy Brewster's Maidstone home in July.
The cat, nicknamed Marmie, was found nearly 40 miles away in Surrey in November after Ms Brewster launched an online media campaign.
Jonathan Brewster was found not guilty of theft at a hearing at Sevenoaks Magistrates' Court.
Speaking outside the court, Ms Brewster said: "I'm just glad to get Marmalade back, that's all that matters at the end of the day."

A BfV spokesman did not confirm a report in Die Welt newspaper that the man was suspected of planning a bomb attack on the BfV's Cologne office, Reuters news agency said.
"There is no evidence to date that there is a concrete danger," he added.
The man is reported to be of Spanish origin and a convert to Islam.
The BfV said the suspect, who now has German citizenship, had previously "behaved inconspicuously".
"The man is accused of making Islamist statements on the Internet using a false name and of revealing internal agency material in Internet chatrooms," the spokesman added.
Die Welt also reported that the man was caught by an agency informant, with the pair having online conversations about a possible attack.
BfV did not confirm which part of the intelligence agency the accused worked in, though German news magazine Der Spiegel said the man was recently hired to observe the Islamist situation in Germany.
It also reported that the man's family had no knowledge of his conversion, which the publication said took place in 2014.
The BfV - the Federal Office for the Protection of the Constitution - is Germany's domestic intelligence agency. Its counterpart, the Federal Intelligence Service (BND), deals with foreign intelligence.
There are an estimated 40,000 Islamists in Germany, including 9,200 ultra-conservative Islamists known as Salafists, the head of the BfV told Reuters news agency earlier in November.
Nursing leaders are warning the NHS in Scotland has "too few nurses" after vacancies rose to record levels.
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A man has been found not guilty of kidnapping his ex-partner's pet cat.
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An employee of the German intelligence agency (BfV) has been arrested after making Islamist statements and sharing agency material, German media report.