Employed in a federally regulated industry, looking to pursue a constructive dismissal case/human rights complaint/Canada labour code complaint due to workplace harassment (toxic environment, excessive unpaid hours, no overtime/lieu time, harassment, sexism). Can I pursue without witnesses?

A legal case against the employer requires evidence as the onus is on you to prove that you've been wronged and suffered damages. You can give evidence and be your own witness, and provide records that support you case in the form of complaints, correspondence, pay statements, schedules, logs of hours worked, etc. Having witnesses to corroborate your evidence is beneficial but a case can be viable even without witnesses, depending on the other evidence you have.