St John Ambulance officers who ditch their uniforms as part of ongoing strike action will have their pay docked by 10 per cent.
The First Union, which represents 1000 ambulance officers across the country, has condemned the move as "astounding".
But St John says it didn't take the step lightly, and it was done out of concern for the health and safety of staff and patients.
The wage deductions come as ambulance officers enter their third month of industrial action, following stalled collective agreement negotiations with St John.
Striking workers are continuing to respond to emergencies and call-outs as normal, but are breaching St John policy by refusing to wear uniforms.
Instead, unionised St John workers have been wearing T-shirts reading "Healthy Ambos Save Lives".
First Union spokesman Jared Abbott said they were taking action to draw the public's attention to issues like growing workloads.
"The wage deductions are pretty astounding. The actions ambulance officers are taking cost St John nothing."
He rejected St John's claim that the deductions were for health and safety reasons, and said members were wearing hi-vis clothing and uniforms when and where appropriate.
"If St John is worried about health and safety then they wouldn't force ambulance officers to work nine hours without a break and they wouldn't send single-crewed ambulances to emergencies," Abbott said.
"It looks like St John is just trying to profit off its own workforce. St John should be offering a pay rise for the hard work ambulance officers are doing, not making a 10 per cent deduction."
St John clinical operations director Norma Lane said ambulance officers needed to be easily recognisable to the public and other emergency service personnel.
It had amended its uniform policy to enable employees to wear the First Union T-shirts, but had also requested staff wear hi-vis vests over the shirts, and proper uniforms on their lower half.
"It is important ambulance officers are identifiable in an emergency environment where circumstances can change rapidly," Lane said.
"Not complying is a health and safety risk not only to the employee but to fellow officers and other emergency workers.
"While there is only a very small number of ambulance professionals refusing to wear hi-vis vests, we have advised First Union and our staff that those employees not complying with this requirement will receive a 10 per cent deduction of wages."
The move was legal under the Employment Relations Act, which enables employers to deduct pay from partial strikers.
"I can assure you we have not taken this step lightly but do so out of concern for the health and safety of our staff as well as for our patients and other emergency personnel who need to be able to be easily identifiable as ambulance personnel," Lane said.
St John had reached agreement with four of the five unions who represent ambulance officers last year. Only the First Union has not reached an agreement.