The youth organisation released a statement yesterday (Tuesday) saying its decision to admit children and adults who identify as women does not put other female members at risk.
However, a campaign group has claimed young people are at risk of being raped or falling pregnant if it continues to admit transgender people.
Girlguiding has been a girl-only organisation since its foundation in 1910, but changed its rules in January 2017 so that any child or young person aged from five to 25 years who self-identifies as a girl or young woman can now become a member regardless of the sex they were assigned at birth.
Helen Watts, one of the expelled volunteers, who has been backed by women's rights campaign group Fair Play For Women, had complained publicly about the policy since it was announced.
In April she and 11 other guide leaders signed a letter to The Sunday Times asking for a review of the policy. Since then, the number of signatories has risen to more than 900.
A Fair Play for Women spokeswoman told CYP Now Girlguiding UK is ignoring basic safeguarding principles with the policy, which says leaders are not allowed to tell girls or their parents if another girl or leader in their unit used to identify as male.
Transgender members are treated the same as other members, and are able to share bedrooms and bathrooms.
"Girlguides have to acknowledge that transgender girls are not the same as female girls," said the spokeswoman.
"The issue is that a transgirl, who is in every respect a male child, is treated as if they were actually a female child. They're not."
She said although numbers of transgender members to have joined the organisation since the policy came into force was unknown, and no safeguarding incidents had been publicly reported as a result, it was "obvious" an issue could arise.
"This is about preventing something from happening and it being obvious that something could happen," she said.
"You mix males and female children - at some point a female child will either get pregnant, raped or, even with consenting exploration, they may do something they regret, or that their parents wished hadn't happened."
She has called upon Girlguiding UK to review the policy, and be more transparent with parents.
In a statement, Girlguiding UK chief guide Amanda Medler and acting chief executive Ruth Marvel said being transgender "does not make someone more of a safeguarding risk than any other person".
"In the last few days you may have seen that Girlguiding's equality and diversity policy has been criticised in the media, with accusations that our inclusion of trans members puts girls at risk. It does not," they said.
"The safety, wellbeing and happiness of our members is at the heart of everything we do in Girlguiding and has been for over 100 years."
They explained the decision to expel two volunteers was taken because of breaches of the organisation's policies, procedures and Volunteer Code of Conduct following an independent investigation based on complaints about the pair.
Girls' Brigade Ministries director Julie Murdy declined to comment on the particular case, but said gender identity is "a significant issue for all uniformed and non-uniformed youth groups", adding that her organisation is continually exploring its approach.
"When dealing with a transgender young person or adult we'd always ask our volunteer leaders to consult with them regarding their specific needs and ensuring we respect their privacy," she said.
Girlguiding UK said it does not hold data on how many transgender members it has, due to data protection regulations.