About 30 people were taking part in the demonstration, holding rainbow flags and banners with slogans including "Transphobia must be stopped" and "If you stay silent, they will come after you too".
But far-right protesters lit smoke bombs and threw them into the crowd, an AFP journalist at the scene said.
The two women attacked with pepper spray were given first aid at the scene, according to the correspondent.
Transgender activists, allies rejoice as historic 'bill of rights' gets parliament's nod
Police led the activists into a subway station and blocked the entrance to protesters to avoid further clashes.
"Today's events have demonstrated that the level far-right radical aggression and violence is increasing in Ukraine," the organisers of the march wrote on Facebook.
Canadian journalist Michael Colborne posted on Twitter that he had been punched in the face by a far-right radical while covering the rally.
'More effective work required on ground for transgender rights'
Homophobia is still commonplace in post-Soviet Ukraine even if Kiev authorities have allowed gay pride marches to take place, in contrast to neighbouring Russia.
Transgender rights marches have taken place in previous years.
Police detained more than 50 far-right activists who tried to disrupt a gay pride march in Kiev this summer.
Around 5,000 people took part in that event amid heightened security.
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