Lib Dem leader Jo Swinson has warned Boris Johnson that "if he thinks being a woman is somehow a weakness, he's about to find out it is not".
She said the PM's choice of insults such as "big girl's blouse" and "girly swot" were "revealing".
In her first conference speech as leader, she said she could not wait to "take on" Mr Johnson, Jeremy Corbyn and Nigel Farage in an election.
And she vowed a Lib Dem government would stop Brexit "on day one".
The Lib Dems currently have 18 MPs - a figure boosted by recent defections - but it would require a seismic shift in the electoral landscape for them to win power.
Nevertheless, Ms Swinson received a standing ovation when she told the conference she wanted to be prime minister, adding: "There is no limit to my ambition for our party."
Mr Johnson called Mr Corbyn "a big girl's blouse" during their first clash at Prime Minister's Questions earlier this month - a remark that prompted some criticism.
On Tuesday, as the Supreme Court began hearing two appeals relating to the suspension of Parliament, Lib Dem conference delegates backed an emergency motion calling for the suspension of Parliament to be reversed.
Earlier at the conference they voted overwhelmingly to back her proposal for a manifesto pledge to revoke Article 50 if the party came into power with a majority government.
In her speech, Ms Swinson criticised Mr Johnson's pledge to take the UK out of the EU by 31 October, with or without a deal.
She told the conference in Bournemouth the prime minister's spending on no-deal preparations was "sickening".
"The truth is you can't plan for no deal. Planning for no deal is like planning to burn your house down," she said. "You might have insurance, but you're still going to lose all your stuff."
Analysis: By Jonathan Blake, BBC political correspondent, in Bournemouth
Ask Liberal Democrat members here what they think of their leader and words like "refreshing", "energetic" and "relatable" trip off the tongue.
Some praise Jo Swinson's ability to communicate with voters, others gush about her confidence and composure in the House of Commons.
But old hands who've seen leaders come and go sound a note of caution about fulfilling expectations.
One senior figure said she needed to "rise to the occasion".
Jo Swinson has won her party's backing for a bold shift in policy on Brexit, and talks of winning 300 seats in a general election.
But with big ambitions come big expectations and soon Jo Swinson will be judged by her party on what she can deliver not just on what she can promise.
Ms Swinson, who succeeded Sir Vince Cable as Lib Dem leader in July, added: "The first task is clear. We must stop Brexit. There is no Brexit that will be good for our country."
She criticised Mr Johnson for withdrawing the Conservative whip from 21 Tory rebels - including one, Sam Gyimah, who later joined the Lib Dems - and for deciding to suspend Parliament.
She said he was "silencing critics, purging opponents, ignoring the law".
"For someone who proclaims to hate socialist dictators, he's doing a pretty good impression of one."
Ms Swinson also turned her fire on Labour leader Jeremy Corbyn, accusing him of holding on to Eurosceptic views.
"Even now, when faced with all the clear and obvious dangers that Brexit brings, Jeremy Corbyn still insists that if Labour win a general election, they will negotiate their own Brexit deal to take us out of the EU," she said.
"Nigel Farage might be Brexit by name, but it is very clear that Jeremy Corbyn is Brexit by nature."
Earlier, shadow attorney general Shami Chakrabarti described the Lib Dem's promise to revoke Article 50 without a further referendum as "illiberal and anti-democratic".
Turning to Scotland, Ms Swinson highlighted its support to remain in the EU in the 2016 referendum and urged supporters there to give "a big vote" to her party.
"Together we can stop Brexit," she said. "We are building a movement across the United Kingdom that is on the verge of stopping it."
'No Planet B'
In her keynote speech, Ms Swinson also touched on policy matters away from Brexit.
On climate change, she said a Lib Dem government would create a green investment bank and set up a citizens' assembly to debate how the UK would reach net-zero carbon emissions by 2045.
She said her government would tackle climate change "because, as the placards say, there is no Planet B."
Ms Swinson also said she wanted the party to "fundamentally rethink the purpose of our economy", asking why a country's success was "reduced to a GDP figure".
"It [GDP] measures everything in short, except that which makes life worthwhile," she said quoting the American politician Bobby Kennedy.
She pledged to introduce a wellbeing budget "to spell out our priorities for public spending on the things that matter most".
The Lib Dem leader also promised to fund youth services in order to tackle knife crime and to ringfence funding for mental health services.