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 £8.4m more on nursing and midwifery bank and agency staff, paying out a total of £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 £450,000 over three years to reintroduce a national return to practice scheme."